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Products _amp; Services

VIEWS: 38 PAGES: 106

									                                    Your partner for customized
                                    screening solutions




Products & Services
Understand how your protein works
Dear researcher,

Thank you for your interest in the services and
products of Dualsystems Biotech.

Dualsystems Biotech develops and markets a range of innovative     While browsing through our catalogue, you will find a range of
technologies in the area of protein interactions, proteomics and   useful research tools, including:
protein expression and characterization.
                                                                   • Services and products for protein interaction discovery
Many of our technologies are provided both as products and         • cDNA library construction services and products
as custom services and their daily application in our laboratory   • Products for protein expression, extraction and refolding
facilities in Zurich, Switzerland enables us to keep abreast of    • Services for profiling the target spectrum of small
technology development and to continuously improve the quality       molecule drugs
of our products.                                                   • Stable cell line generation services

                                                                   We are convinced that our technologies and reagents will help
                                                                   you to stay innovative and competitive in your daily research.

                                                                   Your Dualsystems Biotech Team
Contents
                                                                  1.4 Yeast reporter strains                                 47
1. Protein interaction discovery                          5
                                                                  1.5 Yeast kits and reagents                                48
  Cytosolic and nuclear proteins
                                                                      1.5.1 DS Yeast transformation kit                      48
   1.1 The DUALhybrid (yeast two-hybrid) system           6
                                                                      1.5.2 Single-stranded carrier DNA                      49
        1.1.1 DUALhybrid screening services               8
                                                                      1.5.3 HTX beta-galactosidase assay kit                 50
        1.1.2 DUALhybrid kit                             12
                                                                      1.5.4 SpeedPrep yeast plasmid isolation kit            51
        1.1.3 DUALhybrid vectors                         14
                                                                      1.5.5 Lytic Agent                                      52
        1.1.4 DUALhybrid cDNA libraries                  15
                                                                      1.5.6 Antibodies                                       53
  Membrane proteins
   1.2 The DUALmembrane system                           16           1.5.7 Media packages                                   54

        1.2.1 DUALmembrane screening services            18   2. Drug profiling services
        1.2.2 DUALmembrane starter kits                  22        2 Drug profiling services                                 55

        1.2.3 DUALmembrane pairwise interaction kit      30   3. cDNA library construction
                                                                   3.1 cDNA library construction                             58
        1.2.4 DUALmembrane vectors                       32
                                                                      3.1.1 Normalized cDNA libraries                        60
        1.2.5 DUALmembrane vector complementation sets   34
                                                                      3.1.2 cDNA library construction services               62
        1.2.6 DUALmembrane cDNA libraries                36
                                                                      3.1.3 EasyClone cDNA library construction kits         63
  Nuclear proteins and transcription factors
   1.3 The DUALhunter system                             37
        1.3.1 DUALhunter screening services              39
        1.3.2 DUALhunter starter kits                    43
        1.3.3 DUALhunter vectors                         45                                     Technolgy / general information
        1.3.4 DUALhunter cDNA libraries                  46                                     Custom screening service
                                                                                                Product
4. Protein interaction and activity assays                  6. Protein extraction
    4.1 In vitro binding assays                                  6.1 Extraction from mammalian cells
       4.1.1 Multiwell protein binding plates          65           6.1.1 DUALXtract mammalian cell total protein extraction reagent   82
    4.2 Immunoprecipitation assays                                  6.1.2 DUALXtract total membrane protein extraction kit             83
       4.2.1 FleX-IP immunoprecipitation kit           66           6.1.3 DUALXtract membrane protein optimization kits                84
    4.3 Protein activity assays                                     6.1.4 DUALXtract nuclear and cytoplasmic protein extraction kit    85
       4.3.1 Universal kinase assay kit                67        6.2 Extraction from fungal and bacterial cells
       4.3.2 PARP-1 assay kit                          68           6.2.1 TOTAL protein extraction kit                                 86
5. Protein expression                                            6.3 Protein concentration measurement
    5.1 Expression in E. coli                                       6.3.1 TOTAL protein assay reagent                                  87
       5.1.1 DUALXpress system                         69
                                                            7. Protein refolding
       5.1.2 QuickLyse™ system                         71        7.1 DUALrefold kits
       5.1.3 Expression vectors for Escherichia coli   72           7.1.1 DUALrefold soluble protein refolding kits                    88
    5.2 Expression in yeast                                         7.1.2 DUALrefold membrane protein refolding kits                   89
       5.2.1 KickStart expression system               73        7.2 Protein stability and aggregation assays
       5.2.2 Yeast expression vectors                  75           7.2.1 Protein stability and aggregation assay kit                  90
       5.2.3 Yeast expression strains                  78   8. Kits and reagents for sample preparation
    5.3 Expression in mammalian cells                               8.1.1 QuickSpin protein conc./ buffer exchange/ desalting kit      91
       5.3.1 Stable cell line generation services      79           8.1.2 QuickSpin detergent removal kit                              92

       5.3.2 Mammalian expression vectors              81   9.   Literature                                                            93
                                                            10. License information                                                    97
                                                            11. Shipping information                                                   98
                                                            12. Alphabetical product listing                                           99
                                                                                                                       Protein interaction discovery       1




 Protein interaction discovery
 ›› Identify binding partners for any class of proteins


Applications                                                                   Our screening technologies cover all protein classes
 •• Discover•novel•interaction•partners•of•your•protein•of•interest
                                                                                                                                   Custom•service
 •• Map•protein•interaction•surfaces                                                          Cytosolic       DUALhybrid              page 8
 •• • creen•cytosolic•proteins,•nuclear•proteins•or•integral•
    S                                                                                         & nuclear       (yeast two-hybrid)   Kits
• membrane•proteins                                                                           proteins        system
                                                                                                                                      page 12
 •• All•technologies•are•available•as•custom•services•and•as•kits•

                                                                                                                                   Custom•service
Benefits                                                                       Your protein   Membrane        DUALmembrane            page 18
                                                                               of interest    proteins        system               Kits
•• Screening•technologies•for•all•protein•classes•                                                                                    page 22
•• Protein•interactions•are•detected•in situ•inside•intact•cells
•• • ormalized• cDNA• libraries• ensure• that• even• rare• interactors• are•
   N                                                                                                                               Custom•service
                                                                                              Nuclear
   identified                                                                                 proteins &                              page 39
                                                                                                              DUALhunter
•• No•need•for•protein•extraction•or•purification                                             transcription   system               Kits
•• Simple•transfer•of•interactors•to•downstream•validation•assays                             factors                                 page 43



                                                                                                              Confirmation assays Kits
                                                                                                              Protein binding plates page 65
                                                                                                              FlexIP™ kit            page 66




                                                                                                                                                       5
1.1      Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The DUALhybrid (yeast two-hybrid) system




         The DUALhybrid (yeast two-hybrid) system
         ›› Powerful in situ screening system to identify novel protein interactions


      Applications                                                                     The prey

      •• Discovery•of•novel•interactors•of•your•protein•of•interest                                                                  Gal4
                                                                                                                            Prey
      •• Pairwise•interaction•assays•between•defined•proteins
      •• Domain•mapping•studies
                                                                                                                          OFF
      Benefits                                                                         A•prey•is•constructed•by•fusing•a•second•protein•of•interest•to•the•activa-
      •• In situ•screening•system•–•interactions•are•identified•in•intact•cells        tion•domain•of•a•transcription•factor.
      •• No•optimization•or•washing•steps•needed
      •• Discovery•of•novel•protein•interactions•by•cDNA•library•screening             Interaction between bait and prey


                                                                                                                        Bait Prey     Gal4
      The DUALhybrid technology                                                                               LexA
      The bait

                                       Bait
                             LexA
                                                                                       An•interaction•between•the•two•proteins•recruits•the•prey•to•the•start•
                                                                                       of•the•reporter•gene•and•the•hybrid•transcription•factor•assembled•in•
                                         OFF                                           this•way•from•bait•and•prey•(hence•“two-hybrid”)•activates•the•reporter•
                                                                                       gene.•A•protein•interaction•is•thus•measured•by•reporter•gene•activity.•
      A•bait•is•constructed•by•fusing•a•protein•of•interest•to•the•LexA•domain.•       Commonly•used•reporter•genes•are•either•auxotrophic•markers•which•
      The•LexA•domain•localizes•the•bait•to•a•particular•DNA•sequence•up-              allow•selection•on•a•minimal•medium•or•color•reporters•like•lacZ.
      stream•of•a•reporter•gene.


 6
                                         Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The DUALhybrid (yeast two-hybrid) system                  1.1




Screening of cDNA libraries to identify novel interactions                       What’s a false positive?
The•defined•prey•can•be•replaced•by•a•collection•of•different•preys•(i.e.•       False•positive•is•a•term•for•an•interaction•which•is•identified•in•a•yeast•two-
different•proteins•fused•to•the•activation•domain)•expressed•from•a•cDNA•        hybrid•screen•and•which•cannot•be•reproduced•in•another•experimental•
library.•Each•yeast•cell•expresses•the•same•bait•and•a•particular•prey•from•     system•(e.g.•co-IP,•pulldown,•etc.).•False•positives•include•sticky•proteins,•
the•cDNA•library.•If•bait•and•prey•interact,•the•yeast•cell•multiplies•and•      such•as•chaperones,•partially•folded•proteins•and•promiscous•interactors.
gives•rise•to•a•visible•colony•on•minimal•medium.•All•yeast•cells•which•do•      There•are•several•approaches•to•avoiding•false•positives:
not•express•an•interacting•bait/prey•pair•fail•to•grow•and•are•discarded.•
                                                                                 •• •Use•of•normalized•cDNA•libraries
Finally,•yeast•colonies•are•picked•and•the•prey•plasmid•is•isolated•and•
                                                                                 •• •Optimization•of•selection•conditions
sequenced,•yielding•the•information•on•the•protein•which•interacts•with•
                                                                                 •• •Filtering•of•results•against•false•positive•databases
the•bait.•The•final•result•of•a•yeast•two-hybrid•screen•is•a•list•of•proteins•
which•interact•with•your•protein•of•interest•(the•bait).                         In•general,•when•using•normalized•cDNA•libraries,•carefully•optimized•
                                                                                 selection• conditions• and• database• filtering,• false• positives• are• not• a•
Normalized cDNA libraries                                                        problem•in•yeast•two-hybrid•screening.

The•use•of•normalized•cDNA•libraries•in•yeast•two-hybrid•screening•greatly• Further reading
decreases•the•rate•of•non-specific•interactors•and•increases•chances•
of•identifying•rare•interactors.•Please•see• page 60•for•a•description• •• Ahnesorg•et•al.•Cell•124•(2),•301-313•(2006)
of•normalized•cDNA•libraries.                                               •• Fashena•et•al.•Methods•Enzymol.•328,•14-26•(2000)
                                                                            •• Fields•and•Song•Nature.•340,•245-6•(1989)

                                                                                 The yeast two-hybrid technology is available as custom screen-
                                                                                 ing service and as a kit.
                                                                                 •• Please•see• •page 8•for•custom•screening•services•
                                                                                 •• Please•see• •page 12•for•kits

                                                                                                                                                                    7
1.1.1        Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid screening services




             DUALhybrid screening services
             ›› Highly optimized yeast two-hybrid technology for the identification of specific protein interactions


        Applications                                                                     Save time and money

        •• Identify•novel•interactors•of•your•protein•of•interest                         •• •Results•are•ready•on•average•in•92•days•after•the•start•of•the•screen
        •• Characterize•binary•protein•interactions                                       •• •No•need•to•set•up•yeast•two-hybrid•in•your•lab
        •• Define•binding•motifs•by•domain•mapping                                        •• •Best•value•for•money•in•the•two-hybrid•screening•market

                                                                                         We help you minimize risks
        Benefits
                                                                                          •• •Recloning•of•baits•which•fail•in•the•functional•assay•at•no•charge
        ••   •High•complexity•normalized•cDNA•libraries                                   •• •• escreening•of•a•second•cDNA•library•if•the•first•screen•yields•fewer•
                                                                                              R
        ••   •Exhaustive•screening•using•LiquiMate™•technology                                than•ten•primary•clones•at•no•charge
        ••   •Delivery•of•30•interactors•per•screen•on•average                            •• •• ption•to•switch•to•our•proprietary•DUALhunter•platform•for•strongly•
                                                                                              O
        ••   •Flagging•of•sticky•proteins                                                     self-activating•baits
        ••   •98%•of•all•screens•yield•interactors
                                                                                         Compare us to our competitors – only Dualsystems offers reliable,
        What makes our system the best on the market?                                    high specificity yeast two-hybrid services!

        A commitment to advance your science                                              ••   •Number•of•screens•performed:•>•1’000
                                                                                          ••   •Accumulated•success•rate:•98%
        Like•yourself,•we•are•researchers•and•we•understand•the•importance•
                                                                                          ••   •Screens•yielding•no•interactors:••less•than•1%
        of•your•work.•In•order•for•you•to•gain•the•best•possible•information•from•
                                                                                          ••   •Customer•satisfaction:•95%
        your•yeast•two-hybrid•experiment,•we•carefully•plan•the•screening•with•
        you•and•help•you•overcome•hurdles•in•bait•design,•library•selection•and•
        interpretation•of•results.




  8
       Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid screening services            1.1.1




Service procedure                                                            The•bait•is•screened•against•a•cDNA•library•of•your•choice
                                                                             using•either•high•efficiency•mating•or•sequential•transformation
Part I: Bait construction and analysis




                                                                                                  Mating           Selection
   Bait construction     Yeast transformation      Functional assay
                                                                                                                               Bioinformatic analysis

Advantages
•• •• -•and•C-terminal•bait•vectors•to•decrease•frequency•of•self-
    N                                                                        Data filtering and clone ranking
    activating•baits
                                                                                                      Using•sophisticated•bioinformatic•analysis•tools•
•• •• ighly•sensitive•functional•assay•to•verify•bait•expression•and•
    H
                                                                                                      we• compare• your• screening• results• with• a•
    localization
                                                                                                      comprehensive•background•database•to•elim-
                                                                                                      inate•highly•connected•false•positive•interactors.•
Part II: Library screen and clone analysis
                                                                                                      All• interactors• are• ranked• according• to• the•
•• • ptimization•of•screening•conditions:•the•bait•is•carefully•optimized•
   O                                                                                                  quality•of•the•interaction•(confidence•level).
   using•a•series•of•pilot•screens


        Library



                             Pilot screen              3-AT titration
       Bait strain

                                                                                                                                                            9
1.1.1      Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid screening services




        Advantages                                                                        Human cell lines
        •• • riple•yeast•reporter•strain•(HIS3,•ADE2,•LacZ)•to•decrease•false•
           T                                                                              normalized•HeLa•S3•cells•I•hepatocellular•carcinoma•cells•I•keratinocytes•I•
           positive•rates                                                                 LNCaP•prostate•cancer•cells
        •• • igh• efficiency• LiquiMate™• mating• guarantees• complete• cDNA•
           H
                                                                                          Mouse tissue
           library•coverage•(>•10•x•library•complexity)
        •• • ormalized•cDNA•libraries•decrease•false•positive•rates•and•enable•
           N                                                                              normalized•universal•I•normalized•brain•I•normalized•embryonic•stem•cell•I•
           detection•of•rare•interactors                                                  brain•I•embryo•11•days•I•embryo•17•days•I•testis•
        •• • emi-quantitative•liquid•LacZ•assay•to•determine•the•strength•of•
           S
           an•interaction                                                                 Microorganisms
                                                                                          E.coli:•genomic•library
        Deliverables
                                                                                          Model organisms
        •• 5’•end•sequences•of•all•positive•clones
                                                                                          D. melanogaster:•normalized•universal•I•whole•animal•I•whole•embryo
        •• • lone•analysis,•including•BLAST•search•results•and•clone•grouping•
           C
                                                                                          A. thaliana:•normalized•universal•I•mixed•tissues•I•green•vegetative•tissue
           according•to•confidence•level
                                                                                          C. elegans:•whole•animal
        •• Complete•report
                                                                                          S. cerevisiae:•whole•organism
        •• Optional: delivery of purified plasmid DNA of selected interactors
                                                                                          Rat
        List of all cDNA libraries                                                        liver

        Human tissue                                                                      Normalized

        normalized• universal•I• normalized• brain•I• fetal• brain• I• bone• marrow• I•   human• universal• I• human• brain• I• HeLa• S3• cells• I• mouse• universal• I•
        colon•I•heart•I•kidney•I•leukocytes•I•liver•I•lymph•node•I•ovary•I•pancreas I•    mouse•brain•I•mouse•embryonic•stem•cell•I•D. melanogaster•universal I•
        placenta•I•skeletal•muscle•I•small•intestine•I•spleen•I•testis•                   A. thaliana•universal


  10
Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid screening services              1.1.1




Ordering information: Screening services                                    Pairwise interaction test
                    DUALhybrid•     DUALhybrid•       DUALhybrid•           •• • airwise•interaction•assay•to•determine•whether•two•proteins•in-
                                                                               P
                    Silver•         Gold              Platinum                 teract•directly
 Order•number       S012            S013              S014                  •• Interaction•domain•mapping
 Bait•cloning                                                               •• • omparative• interaction• studies• between• wild• type• and• mutant•
                                                                               C
                                                                               variants•of•a•protein
 Functional•assay
                                                                            •• Fast•and•simple•service
 Library•screen                                                             •• Up•to•96•interactions•tested•in•parallel
 Clone•picking

 Clone•analysis                                                             Ordering information: Additional services

                                                                             Order•number       Service
Additional services                                                          S01101             Bait•dependency•test•(10•interactions)

Bait dependency test                                                         S01102             Pairwise•interaction•test•(10•interactions)

All•prey•clones•of•interest•are•isolated•and•re-transformed•into•yeast•
together•with•the•original•bait•and•an•unrelated•control•bait.•Only•bait-   Selected publications
dependent•interactions•are•scored•as•positive.•A•bait•dependency•test•      ••   Aaman•et•al.•FASEB J.•24,•2334-2346•(2010)
yields•clear•experimental•confirmation•of•a•specific•protein•interaction.   ••   Kajiwara•et•al.•PLoS ONE.•4(4),•e5071•(2009)
                                                                            ••   Ahnesorg•et•al.•Cell 124(2),•301-313•(2006)
                                                                            ••   Hipp•et•al.•J. Biol. Chem.•279(16),•16503-16510•(2004)




                                                                                                                                                       11
1.1.2    Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid kit




         DUALhybrid kit
         ›› Complete kit for yeast two-hybrid screening


        Applications
        •• • iscovery• of• novel• protein• interactions• by• yeast• two-hybrid••
           D                                                                   •
           screening•of•cDNA•libraries
        •• Analysis•of•direct•protein•interactions
        •• Domain•mapping•studies


        Benefits
                                                                                     Kit contents
        •• •• riple•reporter•strain•ensures•stringent•screens•and•low•false•posi-
            T
                                                                                     Content                                          Size
            tive•rates
        •• •N-•and•C-terminal•bait•vectors•for•optimal•bait•construction             pLexA-N•bait•vector                              5•µg
                                                                                     pLexA-C•bait•vector                              5•µg

        Technology                                                                   pGAD-HA•prey•vector                              5•µg
                                                                                     pY3H•bridge•vector                               5•µg
        DUALhybrid•technology•enables•you•to•screen•a•protein•of•interest•for•
                                                                                     pLexA-p53•control•bait•vector                    5•µg
        interactors•expressed•from•a•cDNA•library•of•interest.•Two•different•bait•
                                                                                     pLexA-laminC•control•bait•vector                 5•µg
        vectors•give•you•the•option•to•express•your•protein•of•interest•either•as•
        N-•or•C-terminal•fusion•to•the•LexA•domain.•The•choice•of•two•orienta-       pACT-large•T•control•prey•vector                 5•µg
        tions•enhances•your•chances•of•finding•all•interactors•for•your•protein•     NMY51•yeast•reporter•strain                      stab•culture
        and•reduces•the•chances•of•self-activation.
        The•triple•reporter•strain•NMY51•allows•high•stringency•selection•and•
        greatly•lowers•the•rate•of•false•positives•in•a•screen.




  12
                       Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid kit   1.1.2




Ordering information

Order•number            Product                         Size
P01004                  DUALhybrid•kit                  1•kit


Accessory products
••   DUALhybrid•cDNA•libraries• •page 15
••   DS•Yeast•transformation•kit• •page 48
••   Single•stranded•carrier•DNA• •page 49
••   HTX•beta-galactosidase•assay•kit• •page 50
••   Media•packages•for•yeast•two-hybrid• •page 54




                                                                                                                                                    13
1.1.3     Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid vectors




          DUALhybrid vectors
          ›› Accessory vectors for yeast two-hybrid experiments


        Applications                                                                pY3H bridge vector
        •• • iscovery• of• novel• protein• interactions• by• yeast• two-hybrid•• •• • ridge•vector•to•express•a•third•protein•for•two-hybrid•experiments.•
           D                                                                        B
           screening•of•cDNA•libraries                                              URA3•marker,•compatible•with•the•DUALhybrid•system.
        •• Analysis•of•direct•protein•interactions
        •• Domain•mapping•studies                                                Control plasmid set
                                                                                    •• • •set•of•positive•and•negative•control•interactions•for•two-hybrid•
                                                                                       A
                                                                                       studies:•pLexA-p53,•pACT2-largeT•and•pLexA-laminC.
        pLexA-N bait vector
        •• • tandard•yeast•two-hybrid•bait•vector•with•ADH1•promoter,•2micron• Ordering information
           S
           (high•copy)•origin•and•TRP1•auxotrophic•marker.•LexA•is•fused•to•
           the•N-terminus•of•the•bait•protein.•                                 Order•number Product                                          Size
                                                                                    P03103           pGAD-HA•prey•vector                      5•µg
        pLexA-C bait vector                                                         P03104           pY3H•bridge•vector                       5•µg
                                                                                    P03105           pLexA-N•bait•vector                      5•µg
        •• • everse•orientation•yeast•two-hybrid•bait•vector•with•ADH1•promoter,•
           R
           2micron•(high•copy)•origin•and•TRP1•auxotrophic•marker.•LexA•is•         P03106           pLexA-C•bait•vector                      5•µg
           fused•to•the•C-terminus•of•the•bait•protein.•                            P03107           Control•plasmid•set                      5•µg•each


        pGAD-HA prey vector
        •• • tandard•yeast•two-hybrid•prey•vector•with•ADH1•promoter,•2micron•
           S
           (high•copy)•origin•and•LEU2•auxotrophic•marker.•The•Gal4•activation•
           domain•is•fused•to•the•N-terminus•of•the•prey•protein.•

  14
           Protein•interaction•discovery•|•Cytosolic•and•nuclear•proteins•|•The•DUALhybrid•(yeast•two-hybrid)•system•|•DUALhybrid cDNA libraries      1.1.4




DUALhybrid cDNA libraries
›› cDNA libraries for the DUALhybrid and compatible yeast two-hybrid systems


Applications                                                                Order•number      cDNA•library                                Size
•• • iscovery• of• novel• protein• interactions• by• yeast• two-hybrid••
   D                                                                        Model•organisms
   screening•of•cDNA•libraries                                              P02105            C. elegans•whole•adult                      200•µg
                                                                            P02108            S. cerevisiae                               200•µg
                                                                            P02109            A. thaliana                                 200•µg
Ordering information
                                                                           Please•see• •page 10•for•a•complete•listing•of•cDNA•libraries•available•
Order•number      cDNA•library                                Size         for•DUALhybrid•services.
Mouse•tissue
P02101            11d•total•embryo                            200•µg
P02107            Adult•heart                                 200•µg
P02401            Normalized•adult•brain                      200•µg
Human•tissue
P02102            Colon                                       200•µg
P02402            Normalized•fetal•brain                      200•µg
Human•cell•line
P02103            HeLa•cell•line                              200•µg
P02104            LNCaP•human•prostate•cancer•cells           200•µg
P02106            Hepatocellular•carcinoma•cells              200•µg
Normalized•
P02401            Normalized•adult•mouse•brain                200•µg
P02402            Normalized•human•fetal•brain                200•µg

                                                                                                                                                      15
1.2      Protein•interaction•discovery•|•Membrane•proteins•|•The DUALmembrane system




        The DUALmembrane system*
        ›› Powerful in situ screening system to identify novel protein interactions involving membrane proteins


      Applications                                                                 Mechanism of the DUALmembrane system
      •• Discovery•of•novel•protein•interactions•involving•membrane•proteins       The bait
      •• Pairwise•interaction•assays•between•membrane•proteins                                                A•bait•is•constructed•by•fusing•a•membrane•pro-
      •• Interaction•domain•mapping                                                      Bait                 tein•of•interest•to•the•C-terminal•half•of•ubiquitin•
                                                                                                              (Cub)• and• a• transcription• factor.• The• bait• is• in-
      Benefits                                                                                                serted•into•the•membranes•of•yeast.•Since•the•
                                                                                                              transcription• factor• is• fused• to• the• membrane•
      •• In situ•screening•system•–•interactions•are•identified•at•the•cellular•
                                                                                                              protein,•it•is•immobilized•at•the•membrane•and•
         membrane                                                                  Cub                        cannot•reach•the•nucleus.•Consequently,•the•re-
      •• No•optimization•or•washing•steps•needed
                                                                                                       VP16   porter•genes•integrated•into•the•yeast•genome•
      •• Discovery•of•novel•protein•interactions•by•cDNA•library•screening
                                                                                          LexA                remain•silent.



                                                                                   The prey
                                                                                                              A•prey•is•constructed•by•fusing•a•protein•of•inter-
                                                                                                Prey          est• (either• a• membrane• protein• or• a• cytosolic•
                                                                                                              protein)•to•the•N-terminal•half•of•ubiquitin•carrying•
                                                                                                              a•I3G•point•mutation•(NubG).•


      *•The•DUALmembrane•system•is•a•proprietary•technology•developed•by•
                                                                                          NubG
      • Dualsystems•Biotech•AG.•The•DUALmembrane•technology•is•protected•
      • by•granted•and•pending•patents•worldwide•-•see• •page 97 for•license•
      • information.
 16
                                                                    Protein•interaction•discovery•|•Membrane•proteins•|•The DUALmembrane system                  1.2




Interaction between bait and prey                                                Screening of cDNA libraries to identify novel interactions
                                                                                 The•defined•prey•can•be•replaced•by•a•collection•of•different•preys•(i.e.•
       Bait Prey                                                                 different•proteins•fused•to•the•activation•domain)•expressed•from•a•cDNA•
                                                                                 library.•Each•yeast•cell•expresses•the•same•bait•and•a•particular•prey•
                                                                                 from•the•cDNA•library.•If•bait•and•prey•interact,•the•yeast•cell•multiplies•
                                                                                 and•gives•rise•to•a•visible•colony•on•minimal•medium.•All•yeast•cells•
                                                           VP16                  which•do•not•express•an•interacting•bait/prey•pair•fail•to•grow•and•are•
                                                   LexA
     Split-Ub                                                                    discarded.•Finally,•yeast•colonies•are•picked•and•the•prey•plasmid•is•
                                                                                 isolated•and•sequenced,•yielding•the•information•on•the•protein•which•
                   VP16
           LexA                                                                  interacts•with•the•bait.•The•final•result•of•a•DUALmembrane•screen•is•
                                                                                 a•list•of•proteins•which•interact•with•your•protein•of•interest•(the•bait).

An•interaction•between•bait•and•prey•results•in•assembly•of•split-ubiquitin•
from• Cub• and• NubG.• The• split-ubiquitin• is• recognized• by• ubiquitin-      Further reading
specific•proteases•present•in•yeast,•resulting•in•proteolytic•cleavage•of•       ••   Paumi•et•al.•Mol. Cell•26(1):15-25•(2007)
the•transcription•factor•from•the•bait.•The•liberated•transcription•factor•      ••   Thaminy•et•al.•Genome Research•13:1744-53•(2003)•
moves• to• the• nucleus• and• activates• the• reporter• genes• in• the• yeast•   ••   Auerbach,•et•al.•Proteomics•2,•611-623•(2002)
genome.•The•protein•interaction•which•takes•place•at•the•membrane•               ••   Stagljar•et•al. Proc. Natl. Acad. Sci.•95,•5187-5192•(1998)
is•thus•converted•into•reporter•gene•activity.•Commonly•used•reporter•
genes•are•either•auxotrophic•markers•which•allow•selection•on•a•minimal•         The DUALmembrane technology is available as custom screen-
medium•or•color•reporters•like•lacZ.                                             ing service and as kits.
                                                                                 •• Please•see• •page 18•for•custom•screening•services•
                                                                                 •• Please•see• •page 22•and• •page 30•for•kits


                                                                                                                                                                17
1.2.1        Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane screening services




             DUALmembrane screening services
             ›› In situ screening for membrane protein interactions


        Applications                                                                 Obtain the best results with membrane proteins

        •• Identify•novel•interactors•of•your•membrane•protein•of•interest           •• • creening•of•full-length•integral•membrane•proteins•avoids•artifacts•
                                                                                        S
        •• Characterize•binary•protein•interactions•between•membrane•proteins           and•increases•recovery•of•real•interactors
        •• Compatible•with•GPCRs,•RTKs,•ion•channels•and•others                      •• Interactions•are•detected•in•their•natural•setting•at•the•cellular•membrane
                                                                                     •• Weak•and•transient•interactions•are•detectable

                                                                                     Save time and money
        Benefits
                                                                                     •• Results•are•ready•on•average•in•92•days•after•the•start•of•the•screen
        ••   Screening•of•full-length•membrane•proteins•in situ                      •• No•need•to•set•up•the•DUALmembrane•system•in•your•lab
        ••   High•complexity•normalized•cDNA•libraries
        ••   Exhaustive•screening•using•LiquiMate™•technology                        We help you minimize risks
        ••   Flagging•of•sticky•proteins                                             •• • escreening•of•a•second•cDNA•library•if•the•first•screen•yields•fewer•
                                                                                        R
        ••   98%•of•all•screens•yield•interactors                                       than•ten•primary•clones•at•no•charge

        What makes our system the best on the market?                                Compare us to our competitors – only Dualsystems offers reliable,
                                                                                     high specificity screening services!
        A commitment to advance your science
                                                                                     ••   Number•of•screens•performed:•>•1’000
        In•order•for•you•to•gain•the•best•possible•information•from•your•DUALmem-
                                                                                     ••   Accumulated•success•rate:•98%
        brane•experiment,•we•carefully•plan•the•screening•with•you•and•help•
                                                                                     ••   Screens•yielding•no•interactors:••less•than•1%
        you•overcome•hurdles•in•bait•design,•library•selection•and•interpretation•
                                                                                     ••   Customer•satisfaction:•95%
        of•results.




  18
                             Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane screening serives                    1.2.1




Service procedure                                                              The•bait•is•screened•against•a•cDNA•library•of•your•choice•using•either•
                                                                               high•efficiency•mating•or•sequential•transformation
Part I: Bait construction and analysis




                                                                               •                      Mating          Selection
    Bait construction     Yeast transformation     Functional assay                                                               Bioinformatic analysis

                                                                               Data filtering and clone ranking
Advantages
                                                                                                               Using•sophisticated•bioinformatic•analysis•
•• Choice•of•bait•vectors•for•all•types•of•membrane•proteins                                                   tools•we•compare•your•screening•results•
•• • ighly•sensitive•functional•assay•to•verify•bait•expression•and•lo-
   H                                                                                                           with•a•comprehensive•background•data-
   calization•to•the•membrane                                                                                  base•to•eliminate•highly•connected•false•
                                                                                                               positive•interactors.•
Part II: Library screen and clone analysis
Optimization•of•screening•conditions:•the•bait•is•carefully•optimized•using•
a•series•of•pilot•screens

                                                                               Advantages
         Library
                                                                                   •• • riple•yeast•reporter•strain•(HIS3,•ADE2,•LacZ)•to•decrease•false•
                                                                                      T
                                                                                      positive•rates
                                                                                   •• • igh• efficiency• LiquiMate™• mating• guarantees• complete• cDNA•
                                                                                      H
                             Pilot screen              3-AT titration
        Bait strain                                                                   library•coverage•(>•10•x•library•complexity)

                                                                                                                                                             19
1.2.1      Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane screening services




        Advantages                                                                            Rat cells I NubG-x
        •• • ormalized•cDNA•libraries•decrease•false•positive•rates•and•enable•
           N                                                                                  neonatal•cardiomyocytes•
           detection•of•rare•interactors                                                      Mouse tissue I NubG-x
        •• • emi-quantitative•liquid•LacZ•assay•to•determine•the•strength•of•
           S                                                                                  adult• whole• brain• I• adult• heart• I• adult• kidney• I• whole• embryo,• 11d• I•
           an•interaction                                                                     normalized•brain

        Deliverables                                                                          Mouse tissue I x-NubG
                                                                                              adult•heart•I•adult•spleen•I•whole•embryo,•17d•I•normalized•whole•embryo,•17d
        •• 5’•end•sequences•of•all•positive•clones
        •• • lone•analysis,•including•BLAST•search•results•and•clone•grouping•
           C                                                                                  Model organisms I NubG-x
           according•to•confidence•level                                                      D. melanogaster: adult,•whole•embryo•(16•hours)•I•
        •• Complete•report                                                                    C. elegans:•adult,•eggs•I•A. thaliana•I•Medicago•nodules•I•S. cerevisiae•
        •• Optional: delivery of purified plasmid DNA of selected interactors •
                                                                                              Model organisms I x-NubG
                                                                                              Medicago•nodules
        Available cDNA libraries
                                                                                              Plant I NubG-x
        Human tissue I NubG-x
                                                                                              A. thaliana•I•Medicago•nodules
        adult•brain•I•embryonal•brain•I•adult•colon•I•adult•liver•I•adult•kidney•I•
        adult•lung•I•normalized•spleen•I•normalized•fetal•brain                               Plant I x-NubG
                                                                                              Medicago•nodules
        Human tissue I x-NubG
        adult•brain•I•adult•kidney                                                            Normalized I NubG-x
                                                                                              Mouse•brain•I•Human•spleen•I•Human•fetal•brain•
        Cell lines I NubG-x
        Jurkat•T•cells,•unst.•I•HeLa•cell•line•I•LNCaP•cell•line•I•mammary•epithelial•cells   Normalized I x-NubG
                                                                                              Mouse•whole•embryo,•17d

  20
                            Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane screening serives     1.2.1




Ordering information: Screening services                                   Ordering information: Additional services

 Order•number     Service                                                  Order•number        Service
 S02110           DUALmembrane•services,•part•I                            S02004              Pairwise•interaction•test•(10•interactions)
 S02111           DUALmembrane•services,•part•II
                                                                           Selected publications
Additional services                                                        ••   Egana,•L.A.•et•al.•J Neurosci.•29(14),•4592-604•(2009)
Pairwise interaction test                                                  ••   Felkl•and•Leube•Neuroscience•156(2):344-352•(2008)
                                                                           ••   Paumi•et•al.•Mol. Cell•26(1):15-25•(2007)
•• • airwise•interaction•assay•to•determine•whether•two•proteins•in-
   P
                                                                           ••   Thaminy•et•al.•Genome Research•13:1744-53•(2003)
   teract•directly
•• Interaction•domain•mapping
•• • omparative• interaction• studies• between• wild• type• and• mutant•
   C
   variants•of•a•protein
•• Fast•and•simple•service
•• Up•to•96•interactions•tested•in•parallel




                                                                                                                                             21
1.2.2      Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits




           DUALmembrane starter kits
           ›› Discovery of protein interactions using integral membrane proteins


        Applications                                                                     How to choose the correct starter kit
                                                                                         To•select•the•appropriate•starter•kit•for•your•protein•of•interest,•use•the•
        •• • iscovery•of•novel•protein•interactions•by•DUALmembrane•screen-
           D
                                                                                         selection•scheme•on•the•next•page.•If•you•plan•to•screen•several•mem-
           ing•of•cDNA•libraries
                                                                                         brane•proteins•with•different•topologies,•please•see•the•DUALmembrane•
        •• • ompatible•with•full-length•integral•membrane•proteins•and•mem-
           C
                                                                                         pairwise•interaction•kit•on• •page 30.
           brane-associated•proteins
        •• Analysis•of•direct•protein•interactions
        •• Domain•mapping•studies


        Benefits
        •• ••dentifies•interactions•involving•integral•membrane•proteins•directly•
            I
            at•the•cellular•membrane
        •• •Screening•of•full-length•membrane•proteins


        Technology
        DUALmembrane•technology•enables•you•to•screen•a•membrane•protein•
        of•interest•for•interactors•expressed•from•a•cDNA•library•of•interest.•
        Full-length• proteins• can• be• used• and• the• interaction• takes• place• at•
        the•cellular•membrane.•Please•see• •page 16•for•a•description•of•the•
        DUALmembrane•technology.




  22
                            Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits   1.2.2




Selecting the correct DUALmembrane starter kit for your protein of interest




                              page 28                             page 24




   page 26                    page 28
                                                                                                                                      23
1.2.2      Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits




        DUALmembrane starter kits N                                            Ordering information
                                                                               You•just•have•to•choose•a•cDNA•library•from•our•collection• —• we•as-
                                                                               semble•the•kit•for•you!•


                                                                                Order•    DUALmembrane•starter•      Order• DUALmembrane•starter•
                                                                                number    kit•N•plus•cDNA•library    number kit•N•plus•cDNA•library
                                                                                Human•tissue•I•NubG-x

        Kit contents                                                            P01209    Embryonal•brain            P01217 Adult•kidney
                                                                                P01210    Adult•colon•               P01221 Adult•lung
         Content                                           Size
                                                                                P01212    Adult•liver•               P01230 Normalized•spleen•
         pBT3-N bait vector                                5•µg
                                                                                P01213    Adult•brain•               P01232 Normalized•fetal•brain
         pPR3-N•prey•vector                                5•µg
                                                                                Human•tissue•I•x-NubG
         pOST1-NubI•control•prey•vector                    5•µg
                                                                                P01205    Adult•kidney               P01218 Adult•brain
         pTSU2-APP•control•bait•vector                     5•µg
                                                                                Human•cell•lines•I•NubG-x
         pNubG-Fe65•control•prey•vector                    5•µg
                                                                                P01202    Jurkat•T,•unst.            P01219 LNCaP
         NMY51•yeast•reporter•strain                       stab•culture
                                                                                P01208    HeLa                       P01223 Mammary•epithelial
         HTX•beta-galactosidase•assay•kit                  1•kit
                                                                                Plant•I•NubG-x
         DS•Yeast•transformation•kit                       1•kit
                                                                                P01227 Medicago•nodules•             P01207 A. thaliana
         cDNA•library•of•your•choice                       60•µg
                                                                                Plant•I•x-NubG
        For•further•DUALmembrane•vector•information,•please•see•• •page 32.     P01226    Medicago•nodules•




  24
                                     Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits          1.2.2




Order•    DUALmembrane•starter•      Order• DUALmembrane•starter•          False positive filtering
number    kit•N•plus•cDNA•library    number kit•N•plus•cDNA•library        Submit•the•sequences•of•your•potential•interacting•proteins•and•we•will•
Rat•cells•I•NubG-x                                                         compare•your•screening•results•with•our•comprehensive•background•
P01224    Neonatal•cardiomyocytes                                          database•to•eliminate•false•positive•interactors.
Mouse•tissue•I•NubG-x
P01201    Adult•whole•brain•         P01225 Whole•embryo,•11d•             Ordering information
P01204    Adult•heart                P01231 Normalized•brain•               Order•Number      Service
P01222    Adult•kidney•                                                     S02130            DUALmembrane•service,•false•positive•filtering
Mouse•tissue•I•x-NubG
P01203    Adult•heart•               P01229 Normalized•whole•embryo,•17d
P01216    Adult•spleen•
Model•organisms•I•NubG-x
P01207    A. thaliana                P01228 S. cerevisiae•
P01211    C. elegans•adult•          P01214 C. elegans•eggs•
P01215 D. melanogaster•whole•        P01220 D. melanogaster whole•
       embryo•(16•hours)•                   adult
                                                                           Accessory products
Normalized•cDNA•libraries•I•NubG-x
P01230 Human•spleen                  P01231 Mouse•brain                    ••   DUALmembrane•cDNA•libraries• •page 36
                                                                           ••   SpeedPrep•yeast•plasmid•isolation•kit• •page 51
P01232 Human•fetal•brain
                                                                           ••   Antibodies• page 53
Normalized•cDNA•libraries•I•x-NubG
                                                                           ••   Media•packages• •page 54
P01229 Mouse•whole•embryo,•17d                                             ••   FleX-IP•immunoprecipitation•kit• •page 66

                                                                                                                                                      25
1.2.2      Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits




        DUALmembrane starter kits SUC                                          Ordering information
                                                                               You•just•have•to•choose•a•cDNA•library•from•our•collection• —• we•as-
                                                                               semble•the•kit•for•you!•


                                                                                Order•    DUALmembrane•starter•kit• Order• DUALmembrane•starter•kit•
                                                                                number    SUC•plus•cDNA•library     number SUC•plus•cDNA•library
                                                                                Human•tissue•I•NubG-x
                                                                                P01309    Embryonal•brain            P01317 Adult•kidney
                                                                                P01310    Adult•colon•               P01321 Adult•lung
        Kit contents                                                            P01312    Adult•liver•               P01330 Normalized•spleen•
         Content                                         Size                   P01313    Adult•brain•               P01332 Normalized•fetal•brain
         pBT3-SUC bait vector                            5•µg                   Human•tissue•I•x-NubG
         pPR3-N•prey•vector                              5•µg                   P01305    Adult•kidney               P01318 Adult•brain
         pOST1-NubI•control•prey•vector                  5•µg                   Human•cell•lines•I•NubG-x
         pTSU2-APP•control•bait•vector                   5•µg                   P01302    Jurkat•T,•unst.            P01319 LNCaP
         pNubG-Fe65•control•prey•vector                  5•µg                   P01308    HeLa                       P01323 Mammary•epithelial
         NMY51•yeast•reporter•strain                     stab•culture           Plant•I•NubG-x
         HTX•beta-galactosidase•assay•kit                1•kit                  P01327 Medicago•nodules              P01307 A. thaliana
         DS•Yeast•transformation•kit                     1•kit                  Plant•I•x-NubG
         cDNA•library•of•your•choice                     60•µg                  P01326    Medicago•nodules

        For•further•DUALmembrane•vector•information,•please•see•• •page 32.

  26
                                    Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits          1.2.2




Order•    DUALmembrane•starter•kit• Order• DUALmembrane•starter•kit•      False positive filtering
number    SUC•plus•cDNA•library     number SUC•plus•cDNA•library          Submit•the•sequences•of•your•potential•interacting•proteins•and•we•will•
Rat•cells•I•NubG-x                                                        compare•your•screening•results•with•our•comprehensive•background•
P01324    Neonatal•cardiomyocytes                                         database•to•eliminate•false•positive•interactors.
Mouse•tissue•I•NubG-x
P01301    Adult•whole•brain•        P01325 Whole•embryo,•11d              Ordering information
P01304    Adult•heart               P01331 Normalized•brain•               Order•Number      Service
P01322    Adult•kidney•                                                    S02130            DUALmembrane•service,•false•positive•filtering
Mouse•tissue•I••x-NubG
P01303    Adult•heart•              P01329 Normalized•whole•embryo,•17d
P01316    Adult•spleen•
Model•organisms•I•NubG-x
P01307    A. thaliana               P01328 S. cerevisiae•
P01311    C. elegans•adult•         P01314 C. elegans•eggs•
P01315 D. melanogaster•whole•       P01320 D. melanogaster whole•
       embryo•(16•hours)•                  adult
                                                                          Accessory products
Normalized•I•NubG-x
P01330 Human•spleen                 P01331 Mouse•brain                    ••   DUALmembrane•cDNA•libraries• •page 36
P01332 Human•fetal•brain                                                  ••   SpeedPrep•yeast•plasmid•isolation•kit• •page 51
                                                                          ••   Antibodies• page 53
Normalized•I•x-NubG
                                                                          ••   Media•packages• •page 54
P01329 Mouse•whole•embryo,•17d
                                                                          ••   FleX-IP•immunoprecipitation•kit• •page 66

                                                                                                                                                     27
1.2.2      Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits




        DUALmembrane starter kits STE                                          Ordering information
                                                                               You•just•have•to•choose•a•cDNA•library•from•our•collection• —• we•as-
                                                                               semble•the•kit•for•you!•


                                                                                Order•    DUALmembrane•starter•       Order• DUALmembrane•starter•
                                                                                number    kit•STE•plus•cDNA•library   number kit•STE•plus•cDNA•library
                                                                                Human•tissue•I•NubG-x
                                                                                P01409    Embryonal•brain             P01417 Adult•kidney
                                                                                P01410    Adult•colon•                P01421 Adult•lung
                                                                                P01412    Adult•liver•                P01430 Normalized•spleen•
         Content                                          Size                  P01413    Adult•brain•                P01432 Normalized•fetal•brain
         pBT3-STE bait vector                             5•µg                  Human•tissue•I•x-NubG
         pPR3-N•prey•vector                               5•µg                  P01405    Adult•kidney                P01418 Adult•brain
         pOST1-NubI•control•prey•vector                   5•µg                  Human•cell•lines•I•NubG-x
         pTSU2-APP•control•bait•vector                    5•µg                  P01402    Jurkat•T                    P01419 LNCaP
         pNubG-Fe65•control•prey•vector                   5•µg                  P01408    HeLa                        P01423 Mammary•epithelial
         NMY51•yeast•reporter•strain                      stab•culture          Plant•I•NubG-x
         HTX•beta-galactosidase•assay•kit                 1•kit                 P01427 Medicago•nodules•              P01407 A. thaliana
         DS•Yeast•transformation•kit                      1•kit                 Plant•I•x-NubG
         cDNA•library•of•your•choice                      60•µg                 P01426    Medicago•nodules

        For•further•DUALmembrane•vector•information,•please•see•• •page 32.


  28
                                      Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane starter kits          1.2.2




Order•    DUALmembrane•starter•       Order• DUALmembrane•starter•          False positive filtering
number    kit•STE•plus•cDNA•library   number kit•STE•plus•cDNA•library      Submit•the•sequences•of•your•potential•interacting•proteins•and•we•will•
Rat•cells•I•NubG-x                                                          compare•your•screening•results•with•our•comprehensive•background•
P01424    Neonatal•cardiomyocytes                                           database•to•eliminate•false•positive•interactors.
Mouse•tissue•I•NubG-x
P01401    Adult•whole•brain•          P01425 Whole•embryo,•11d•             Ordering information
P01404    Adult•heart                 P01431 Normalized•brain•               Order•Number      Service
P01422    Adult•kidney•                                                      S02130            DUALmembrane•service,•false•positive•filtering
Mouse•tissue•I••x-NubG
P01403    Adult•heart•                P01429 Normalized•whole•embryo,•17d
P01416    Adult•spleen•
Model•organisms•I•NubG-x
P01407    A. thaliana                 P01428 S. cerevisiae•
P01411    C. elegans•adult•           P01414 C. elegans•eggs•
P01415 D. melanogaster•whole•         P01420 D. melanogaster whole•
       embryo•(16•hours)•                    adult
                                                                            Accessory products
Normalized•I•NubG-x
P01430 Human•spleen                   P01431 Mouse•brain                    ••   DUALmembrane•cDNA•libraries• •page 36
                                                                            ••   SpeedPrep•yeast•plasmid•isolation•kit• •page 51
P01432 Human•fetal•brain
                                                                            ••   Antibodies• page 53
Normalized•I•x-NubG
                                                                            ••   Media•packages• •page 54
P01429 Mouse•whole•embryo,•17d
                                                                            ••   FleX-IP•immunoprecipitation•kit• •page 66

                                                                                                                                                       29
1.2.3     Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane pairwise interaction kit




          DUALmembrane pairwise interaction kit
          ›› Discovery of protein interactions using integral membrane proteins


        Applications                                                                DUALmembrane pairwise interaction kit
        •• Analysis•of•direct•interactions•involving•membrane•proteins              The•DUALmembrane•pairwise•interaction•kit•contains•all•bait•vectors,•
        •• • iscovery•of•novel•protein•interactions•by•DUALmembrane•screen-
           D                                                                        prey•vectors•and•controls•to•perform•pairwise•interaction•assays•between•
           ing•of•cDNA•libraries                                                    membrane•proteins•of•all•classes.•When•used•in•combination•with•an•
        •• Compatible• with• full-length• integral• membrane• proteins• and••       appropriate•DUALmembrane•cDNA•library,•the•pairwise•interaction•kit•
        • membrane-associated•proteins                                              allows•screening•of•any•membrane•protein•to•identify•novel•interactors.
        •• Domain•mapping•studies



        Benefits
        •• •dentifies•interactions•involving•integral•membrane•proteins•directly•
           I
           at•the•cellular•membrane
        •• Screening•of•full-length•membrane•proteins




  30
                        Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane pairwise interaction kit   1.2.3




Kit contents                                                           Ordering information

 Component                                             Size             Order•number       Product                                   Size
 pBT3-N•bait•vector                                    5•µg             P01501             DUALmembrane•pairwise•interaction•kit     1•kit
 pBT3-C•bait•vector                                    5•µg
 pBT3-SUC•bait•vector                                  5•µg
 pBT3-STE•bait•vector                                  5•µg
 pPR3-N•prey•vector                                    5•µg
 pPR3-C•prey•vector                                    5•µg
 pPR3-SUC•prey•vector                                  5•µg
 pPR3-STE•prey•vector                                  5•µg
 pOST1-NubI•control•prey•vector                        5•µg
 pTSU2-APP•control•bait•vector                         5•µg
 pNubG-Fe65•control•prey•vector                        5•µg            Accessory products
 NMY51•yeast•reporter•strain                           stab•culture
                                                                        ••   DUALmembrane•cDNA•libraries• •page 36
 DS•Yeast•transformation•kit                           1•kit            ••   SpeedPrep•yeast•plasmid•isolation•kit• •page 51
 HTX•beta-galactosidase•assay•kit                      1•kit            ••   Antibodies• page 53
                                                                        ••   Media•packages• •page 54
For•further•DUALmembrane•vector•information,•please•see•• •page 32.     ••   FleX-IP•immunoprecipitation•kit• •page 66




                                                                                                                                              31
1.2.4         Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane vectors




              DUALmembrane vectors
              ›› Vectors for the DUALmembrane system


        Applications                                                              pBT3-STE bait vector
            •• • iscovery•of•novel•protein•interactions•by•DUALmembrane•screen-
               D                                                                      •• • ait•vector•for•membrane•proteins•with•a•cytosolic•C-terminus•and•
                                                                                         B
               ing•of•cDNA•libraries                                                     an•extracellular•N-terminus•or•for•membrane•proteins•with•both•ter-
            •• • ompatible•with•full-length•integral•membrane•proteins•and•mem-
               C                                                                         mini•in•the•cytoplasm.•The•Cub•moiety•is•fused•to•the•C-terminus•
               brane-associated•proteins                                                 of•the•bait•protein.
            •• Analysis•of•direct•protein•interactions
            •• Domain•mapping•studies                                             pBT3-C bait vector
                                                                                      •• • ait•vector•for•yeast•membrane•proteins.•The•Cub•moiety•is•fused•
                                                                                         B
                                                                                         to•the•C-terminus•of•the•bait•protein.
        pBT3-N bait vector
            •• • ait•vector•for•membrane•proteins•with•cytosolic•N-terminus•
               B
                                                                                  pPR3-N prey vector
        •      (type•II•membrane•proteins).•The•Cub•moiety•is•fused•to•the•
        •      N-terminus•of•the•bait•protein.                                        •• Prey•vector•for•membrane•proteins•with•cytosolic•N-terminus•
                                                                                  •      (type•II•membrane•proteins)•and•for•cytosolic•proteins.•The•NubG••
                                                                                  •      moiety•is•fused•to•the•N-terminus•of•the•prey•protein.
        pBT3-SUC bait vector
         •• • ait•vector•for•membrane•proteins•with•a•cytosolic•C-terminus•and•
            B                                                                 •
                                                                                  pPR3-C prey vector
            a•N-terminal•cleavable•signal•sequence•(type•I•membrane•proteins•
            such•as•RTKs•and•GPCRs).•The•Cub•moiety•is•fused•to•the•                  •• Prey•vector•for•yeast•membrane•and•cytosolic•proteins.•The•Cub••
        • C-terminus•of•the•bait•protein.                                             • moiety•is•fused•to•the•C-terminus•of•the•prey•protein.




  32
                                          Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane vectors   1.2.4




pPR3-SUC prey vector                                                         Ordering information
    •• Prey•vector•for•membrane•proteins•with•a•cytosolic•C-terminus•and•
                                                                             Order•number   Product                                  Size
•      a•N-terminal•cleavable•signal•sequence•(type•I•membrane•proteins•
                                                                             P03230         pBT3-N•bait•vector                       5•µg
•      such•as•RTKs•and•GPCRs).•The•NubG•moiety•is•fused•to•the•
•      C-terminus•of•the•prey•protein.                                       P03231         pBT3-C•bait•vector                       5•µg
                                                                             P03232•        pBT3-SUC•bait•vector                     5•µg
                                                                             P03233         pBT3-STE•bait•vector                     5•µg
pPR3-STE prey vector
                                                                             P03234         pPR3-N•prey•vector                       5•µg
    •• • rey•vector•for•membrane•proteins•with•a•cytosolic•C-terminus•and•
       P
       an•extracellular•N-terminus•or•for•membrane•proteins•with•both•ter-   P03235         pPR3-C•prey•vector                       5•µg
       mini•in•the•cytoplasm.•The•NubG•moiety•is•fused•to•the•C-terminus•    P03236         pPR3-SUC•prey•vector                     5•µg
       of•the•prey•protein.                                                  P03237         pPR3-STE•prey•vector                     5•µg
                                                                             P03243•        DUALmembrane•control•plasmid•set         5•µg•
Control plasmid set                                                                                                                  each

    •• • •set•of•positive•and•negative•control•interactions•for•DUALmem-
       A
       brane•studies:•pTSU2-APP•control•bait,•pNubG-Fe65•control•prey,•
       pOST1-NubI•positive•control•prey.




                                                                                                                                               33
1.2.5      Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane vector complementation sets




           DUALmembrane vector complementation sets
           ›› Discovery of protein interactions using integral membrane proteins


        Applications                                                                Choosing the correct vector complementation set
        •• Analysis•of•direct•interactions•involving•membrane•proteins               Starter•kit            Correct•complementation•set
        •• • iscovery•of•novel•protein•interactions•by•DUALmembrane•screen-
           D                                                                         Starter•kit•N          Complementation•set•N
           ing•of•cDNA•libraries
                                                                                     Starter•kit•SUC        Complementation•set•SUC
        •• • ombine•with•a•DUALmembrane•starter•kit•to•screen•any•mem-
           C
                                                                                     Starter•kit•STE        Complementation•set•STE
           brane•protein•of•interest

                                                                                    Kit contents
        Benefits                                                                    DUALmembrane vector complementation set N*
        •• •dentifies•interactions•involving•integral•membrane•proteins•directly•
           I                                                                        *available•only•together•with•the•DUALmembrane•starter•kit•N
           at•the•cellular•membrane                                                  Component                                               Amount
        •• Screening•of•full-length•membrane•proteins                                pBT3-C•bait•vector                                      5•µg
                                                                                     pBT3-SUC•bait•vector                                    5•µg
        DUALmembrane vector complementation sets                                     pBT3-STE•bait•vector                                    5•µg

        Combination•of•a•DUALmembrane•starter•kit•with•the•appropriate•vector•       pPR3-C•prey•vector                                      5•µg
        complementation•set•gives•you•the•full•choice•of•DUALmembrane•bait•          pPR3-SUC•prey•vector                                    5•µg
        vectors•and•allows•you•to•carry•out•screenings•and•pairwise•interaction•     pPR3-STE•prey•vector                                    5•µg
        assays•involving•any•membrane•protein•or•cytosolic•protein.
                                                                                    For•further•DUALmembrane•vector•information,•please•see•• •page 32.




  34
                 Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane vector complementation sets      1.2.5




DUALmembrane vector complementation set SUC*                         Ordering information
*available•only•together•with•the•DUALmembrane•starter•kit•SUC
                                                                      Order•number     Product                                       Size
 Component                                               Amount
                                                                      P01502           DUALmembrane•vector•complementation•set•N     1•set
 pBT3-C•bait•vector                                      5•µg
                                                                      P01503           DUALmembrane•vector•complementation•set•SUC 1•set
 pBT3-N•bait•vector                                      5•µg
                                                                      P01504           DUALmembrane•vector•complementation•set•STE   1•set
 pBT3-STE•bait•vector                                    5•µg
 pPR3-C•prey•vector                                      5•µg
                                                                     Accessory products
 pPR3-SUC•prey•vector                                    5•µg
 pPR3-STE•prey•vector                                    5•µg         ••   DUALmembrane•cDNA•libraries• •page 36
                                                                      ••   DS•Yeast•transformation•kit• •page 48
For•further•DUALmembrane•vector•information,•please•see• •page 32.    ••   Single•stranded•carrier•DNA• page 49
                                                                      ••   HTX•beta-galactosidase•assay•kit• •page 50
DUALmembrane vector complementation set STE*                          ••   Media•packages• •page 54
*available•only•together•with•the•DUALmembrane•starter•kit•STE
 Component                                              Amount
 pBT3-C•bait•vector                                     5•µg
 pBT3-N•bait•vector                                     5•µg
 pBT3-SUC•bait•vector                                   5•µg
 pPR3-C•prey•vector                                     5•µg
 pPR3-SUC•prey•vector                                   5•µg
 pPR3-STE•prey•vector                                   5•µg

For•further•DUALmembrane•vector•information,•please•see• •page 32.

                                                                                                                                             35
1.2.6     Protein•interaction•discovery•|•Membrane•proteins•|•The•DUALmembrane•system•|•DUALmembrane cDNA libraries




          DUALmembrane cDNA libraries
          ›› cDNA libraries for the DUALmembrane system


        Applications                                                          Order•    Product•                  Order• Product•
        •• • iscovery•of•novel•protein•interactions•by•DUALmembrane•screen-
           D                                                                  number    Size:•200•µg              number Size:•200•µg
           ing•of•cDNA•libraries                                              Rat•cells•I•NubG-x
                                                                              P02233    Neonatal•cardiomyocytes
                                                                              Mouse•tissue•I•NubG-x
         Order•    Product•              Order• Product•                      P02201    Adult•whole•brain•        P02234 Whole•embryo,•11d•
         number    Size:•200•µg          number Size:•200•µg                  P02207    Adult•heart               P02303 Normalized•brain•
         Human•tissue•I•NubG-x                                                P02231    Adult•kidney•
         P02213    Embryonal•brain       P02226 Adult•kidney                  Mouse•tissue•I•x-NubG
         P02215    Adult•colon•          P02230 Adult•lung                    P02206    Adult•heart•              P02301 Normalized•whole•embryo,•17d
         P02220    Adult•liver•          P02302 Normalized•spleen•            P02224    Adult•spleen•
         P02221    Adult•brain•          P02304 Normalized•fetal•brain        Model•organisms•I•NubG-x
         Human•tissue•I•x-NubG                                                P02210    A. thaliana               P02237 S. cerevisiae•
         P02208    Adult•kidney          P02227 Adult•brain                   P02218    C. elegans•adult•         P02222 C. elegans•eggs•
         Human•cell•lines•I•NubG-x                                            P02223 D. melanogaster•whole• P02229 D. melanogaster whole•
         P02205    Jurkat•T,•unst.       P02228 LNCaP                                embryo•(16•hours)•            adult
         P02212    HeLa                  P02232 Mammary•epithelial            Normalized•I•NubG-x
         Plant•I•NubG-x                                                       P02302 Human•spleen                 P02303 Mouse•brain
         P02236 Medicago•nodules         P02210 A. thaliana                   P02304 Human•fetal•brain
         Plant•I•x-NubG                                                       Normalized•I•NubG-x
         P02235    Medicago•nodules                                           P02301 Mouse•whole•embryo,•17d


  36
                                                   Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The DUALhunter system              1.3




The DUALhunter system
›› Powerful in situ screening system to identify novel protein interactions involving nuclear proteins, transcription factors and self-activating proteins


Applications                                                                   Mechanism of the DUALhunter system
•• • iscovery•of•novel•protein•interactions•involving•nuclear•proteins,•
   D                                                                           The bait
   transcription•factors•and•acidic•proteins                                              Ost4p          A•bait•is•constructed•by•fusing•a•protein•of•inter-
•• Pairwise•interaction•assays•between•nuclear•proteins                                                  est•at•its•N-terminus•to•a•small•membrane•anchor•
                                                                                                         (Ost4p)•and•at•its•C-terminus•to•the•C-terminal•
Benefits                                                                                                 half•of•ubiquitin•(Cub)•and•a•transcription•factor.•
                                                                                   Bait
                                                                                                         The•bait•is•inserted•into•the•membranes•of•yeast.•
•• In situ•screening•system•–•no•cell•lysis•or•washing•steps•needed
                                                                                                         Since• the• transcription• factor• is• fused• to• the•
•• No•optimization•needed                                                       Cub                      membrane•protein,•it•is•immobilized•at•the•mem-
•• Discovery•of•novel•protein•interactions•by•cDNA•library•screening
                                                                                                  VP16   brane• and• cannot• reach• the• nucleus.• Conse-
                                                                                       LexA              quently,•the•reporter•genes•integrated•into•the•
                                                                                                         yeast•genome•remain•silent.

                                                                               The prey
                                                                                                         A•prey•is•constructed•by•fusing•a•second•protein•
                                                                                                         of•interest••to•the•N-terminal•half•of•ubiquitin•car-
                                                                                                         rying•a•I3G•point•mutation•(NubG).•

                                                                                           Prey


                                                                                           NubG




                                                                                                                                                                  37
1.3      Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The DUALhunter system




      Interaction between bait and prey                                                Screening of cDNA libraries to identify novel interactions
                Ost4p                                                                  The•defined•prey•can•be•replaced•by•a•collection•of•different•preys•(i.e.•
                                                                                       different•proteins•fused•to•the•activation•domain)•expressed•from•a•cDNA•
                                                                                       library.•Each•yeast•cell•expresses•the•same•bait•and•a•particular•prey•from•
                                                                                       the•cDNA•library.•If•bait•and•prey•interact,•the•yeast•cell•multiplies•and•
            Bait Prey
                                                                                       gives•rise•to•a•visible•colony•on•minimal•medium.•All•yeast•cells•which•do•
                                                                  VP16                 not•express•an•interacting•bait/prey•pair•fail•to•grow•and•are•discarded.•
                                                          LexA
            Split-Ub                                                                   Finally,•yeast•colonies•are•picked•and•the•prey•plasmid•is•isolated•and•
                         VP16                                                          sequenced,•yielding•the•information•on•the•protein•which•interacts•with•
                 LexA                                                                  the•bait.•The•final•result•of•a•DUALhunter•screen•is•a•list•of•proteins•that•
                                                                                       interact•with•your•protein•of•interest•(the•bait).

      An•interaction•between•bait•and•prey•results•in•assembly•of•split-ubiquitin•
      from• Cub• and• NubG.• The• split-ubiquitin• is• recognized• by• ubiquitin-      Further reading
      specific•proteases•present•in•yeast,•resulting•in•proteolytic•cleavage•of•       •• Möckli•et•al.•BioTechniques•42(6):725-730•(2007)
      the•transcription•factor•from•the•bait.•The•liberated•transcription•factor•
      moves• to• the• nucleus• and• activates• the• reporter• genes• in• the• yeast•   The DUAhunter technology is available as custom screening
      genome.•The•protein•interaction•is•thus•converted•into•reporter•gene•            services and as kits.
      activity.•Commonly•used•reporter•genes•are•either•auxotrophic•markers•
                                                                                       •• Please•see• •page 39•for•custom•screening•services•
      which•allow•selection•on•a•minimal•medium•or•color•reporters•like•lacZ.
                                                                                       •• Please•see• •page 43•for•kits




 38
                Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter screening services            1.3.1




DUALhunter screening services
›› In situ screening for protein interactions using nuclear proteins, transcription factors and self-activating proteins


Applications                                                                    What makes our system the best on the market?
•• •dentify•novel•interactors•of•nuclear•proteins,•acidic•proteins•and•
   I                                                                            A commitment to advance your science
   transcription•factors                                                        In•order•for•you•to•gain•the•best•possible•information•from•your•DUAL-
•• • creening•of•proteins•which•are•self-activating•in•classical• two-
   S                                                                            hunter•experiment,•we•carefully•plan•the•screening•with•you•and•help•
   hybrid•systems                                                               you•overcome•hurdles•in•bait•design,•library•selection•and•interpretation•
•• Characterize•binary•protein•interactions                                     of•results.

                                                                                Save time and money
Benefits                                                                         •• Results•are•ready•on•average•in•92•days•after•the•start•of•the•screen
                                                                                 •• No•need•to•set•up•the•DUALhunter•system•in•your•lab
••   High•complexity•normalized•cDNA•libraries
••   Exhaustive•screening•using•LiquiMate™•technology
                                                                                We help you minimize risks
••   Flagging•of•sticky•proteins
••   98%•of•all•screens•yield•interactors                                        •• • escreening•of•a•second•cDNA•library•if•the•first•screen•yields•fewer•
                                                                                    R
                                                                                    than•ten•primary•clones•at•no•charge

                                                                                Compare us to our competitors – only Dualsystems offers reliable,
                                                                                high specificity screening services!
                                                                                 ••   Number•of•screens•performed:•>•1’000
                                                                                 ••   Accumulated•success•rate:•98 %
                                                                                 ••   Screens•yielding•no•interactors:•less•than•1 %
                                                                                 ••   Customer•satisfaction:•95 %



                                                                                                                                                              39
1.3.1      Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter screening services




        Service procedure                                                                The•bait•is•screened•against•a•cDNA•library•of•your•choice
                                                                                         using•either•high•efficiency•mating•or•sequential•transformation
        Part I: Bait construction and analysis




                                                                                                                 Mating          Selection
            Bait construction      Yeast transformation      Functional assay            •                                                   Bioinformatic analysis

                                                                                         Data filtering and clone ranking
        Advantages
                                                                                                                  Using•sophisticated•bioinformatic•analysis•tools•
        •• • ighly•sensitive•functional•assay•to•verify•bait•expression•and•lo-
           H                                                                                                      we•compare•your•screening•results•with•a•com-
           calization•to•the•membrane                                                                             prehensive• background• database• to• eliminate•
                                                                                                                  highly•connected•false•positive•interactors.•
        Part II: Library screen and clone analysis
        Optimization• of• screening• conditions:• the• bait• is• carefully• optimized•
        using•a•series•of•pilot•screens                                                  Advantages
                                                                                             •• • riple•yeast•reporter•strain•(HIS3,•ADE2,•LacZ)•to•decrease•false•
                                                                                                T
                 Library                                                                        positive•rates
                                                                                             •• • igh• efficiency• LiquiMate™• mating• guarantees• complete• cDNA•
                                                                                                H
                                                                                                library•coverage•(>•10•x•library•complexity)
                                      Pilot screen               3-AT titration              •• • ormalized•cDNA•libraries•decrease•false•positive•rates•and•enable•
                                                                                                N
                Bait strain
                                                                                                detection•of•rare•interactors

  40
                  Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter screening services                       1.3.1




Advantages                                                                            Mouse tissue I NubG-x
•• • emi-quantitative•liquid•LacZ•assay•to•determine•the•strength•of•
   S                                                                                  adult• whole• brain• I• adult• heart• I• adult• kidney• I• whole• embryo,• 11d• I•
   an•interaction                                                                     normalized•brain

                                                                                      Mouse tissue I x-NubG
Deliverables                                                                          adult•heart•I•adult•spleen•I•whole•embryo,•17d•I•normalized•whole•em-
•• 5’•end•sequences•of•all•positive•clones                                            bryo,•17d
•• • lone•analysis,•including•BLAST•search•results•and•clone•grouping•
   C
                                                                                      Model organisms I NubG-x
   according•to•confidence•level
•• Complete•report                                                                    D. melanogaster: adult,•whole•embryo•(16•hours)•I•
•• Optional: delivery of purified plasmid DNA of selected interactors                 C. elegans:•adult,•eggs•I•
                                                                                      A. thaliana•I•Medicago•nodules•I•S. cerevisiae•

Available cDNA libraries                                                              Model organisms I x-NubG
                                                                                      Medicago•nodules•
Human tissue I NubG-x
adult•brain•I•embryonal•brain•I•adult•colon•I•adult•liver•I•adult•kidney•I•           Plant I NubG-x
adult•lung•I•normalized•spleen•I•normalized•fetal•brain                               A. thaliana•I•Medicago•nodules
Human tissue I x-NubG                                                                 Plant I x-NubG
adult•brain•I•adult•kidney                                                            Medicago•nodules
Cell lines I NubG-x                                                                   Normalized I NubG-x
Jurkat•T•cells,•unst.•I•HeLa•cell•line•I•LNCaP•cell•line•I•mammary•epithelial•cells   Mouse•brain•I•Human•spleen•I•Human•fetal•brain•
Rat cells I NubG-x                                                                    Normalized I x-NubG
neonatal•cardiomyocytes•                                                              Mouse•whole•embryo,•17d

                                                                                                                                                                           41
1.3.1     Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter screening services




        Ordering information: Screening services                                   Selected publications

         Order•number     Service                                                  •• Möckli•et•al.•BioTechniques•42(6):725-730•(2007)
         S05110           DUALhunter•services,•part•I
         S05111           DUALhunter•services,•part•II

        Additional services
        Pairwise interaction test
        •• • airwise•interaction•assay•to•determine•whether•two•proteins•in-
           P
           teract•directly
        •• Interaction•domain•mapping
        •• • omparative• interaction• studies• between• wild• type• and• mutant•
           C
           variants•of•a•protein
        •• Fast•and•simple•service
        •• Up•to•96•interactions•tested•in•parallel


        Ordering information: Additional services

         Order•number    Service
         S05201          DUALhunter•service,•pairwise•interaction•test•
                         (10•interactions)




  42
                          Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter starter kits       1.3.2




DUALhunter starter kits
›› Discovery of protein interactions using nuclear proteins, transcription factors and self-activating proteins


Applications                                                                   DUALhunter starter kits
•• Discovery•of•novel•protein•interactions•by•screening•of•cDNA•libraries      DUALhunter•starter•kits•contain•a•bait•vector,•controls•and•a•cDNA•library•
•• • ompatible•with•nuclear•proteins,•transcription•factors,•acidic•pro-
   C                                                                           of•your•choice.•You•just•have•to•choose•a•cDNA•library•from•our•collection•
   teins•and•self-activating•proteins                                          (see• •page 46)•and•we•assemble•the•kit•for•you!•
•• Analysis•of•direct•protein•interactions
•• Domain•mapping•studies                                                      Kit contents

                                                                                 Content                                              Size
Benefits                                                                         pDHB1•bait•vector                                    5•ug
                                                                                 pPR3-N•prey•vector                                   5•ug
•• • lternative•for•screening•proteins•which•are•self-activating•in•clas-
   A
                                                                                 pOST1-NubI•control•prey•vector                       5•ug
   sical•yeast•two-hybrid•systems
•• Screening•of•acidic•proteins•and•transcription•factors                        pDSL-p53•control•prey•vector                         5•ug
                                                                                 pDHB1-Large•T•control•bait•vector                    5•ug

Technology                                                                       NMY51•yeast•reporter•strain                          stab•culture
                                                                                 HTX•beta-galactosidase•assay•kit                     1•kit
DUALhunter•technology•enables•you•to•screen•proteins•which•are•un-
                                                                                 DS•Yeast•transformation•kit                          1•kit
suitable•for•use•in•classical•two-hybrid•systems,•such•as•transcription•
                                                                                 cDNA•library•of•your•choice                          60•µg
factors,•nuclear•proteins•or•acidic•proteins.•The•bait•protein•is•attached•
to•the•membrane•via•a•small•transmembrane•domain•and•is•screened•
outside•of•the•nucleus•for•interaction•with•other•proteins.•Please•see• •
page 37•for•a•description•of•the•DUALhunter•technology.
•


                                                                                                                                                             43
1.3.2     Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter starter kits




         DUALhunter starter kits
         ›› Discovery of protein interactions using nuclear proteins, transcription factors and self-activating proteins


        Ordering information                                                           Order•     DUALhunter•starter•kit•   Order• DUALhunter•starter•kit•
                                                                                       number     plus•cDNA•library         number plus•cDNA•library
         Order•    DUALhunter•starter•kit•    Order• DUALhunter•starter•kit•
         number    plus•cDNA•library          number plus•cDNA•library                 Rat•cells•I•NubG-x

         Human•tissue•I•NubG-x                                                         P01624     Neonatal•cardiomyocytes

         P01609    Embryonal•brain            P01617 Adult•kidney                      Plant•I•NubG-x

         P01610    Adult•colon•               P01621 Adult•lung                        P01627 Medicago•nodules•             P01607 A. thaliana

         P01612    Adult•liver•               P01630 Normalized•spleen•                Plant•I•x-NubG

         P01613    Adult•brain•               P01632 Normalized•fetal•brain            P01626     Medicago•nodules

         Human•tissue•I•x-NubG                                                         Model•organisms•I•NubG-x

         P01605    Adult•kidney               P01618 Adult•brain                       P01607     A. thaliana               P01628 S. cerevisiae•

         Human•cell•lines•I•NubG-x                                                     P01611     C. elegans•adult•         P01614 C. elegans•eggs•

         P01602    Jurkat•T,•unst.            P01619 LNCaP                             P01615 D. melanogaster•whole•        P01620 D. melanogaster whole
                                                                                              embryo•(16•hours)•                   adult
         P01608    HeLa                       P01623 Mammary•epithelial
                                                                                       Normalized•I•NubG-x
         Mouse•tissue•I•NubG-x
                                                                                       P01630 Human•spleen•                 P01631 Mouse•brain
         P01601    Adult•whole•brain•         P01625 Whole•embryo,•11d
                                                                                       P01632 Human•fetal•brain
         P01604    Adult•heart                P01631 Normalized•brain•
                                                                                       Normalized•I•x-NubG
         P01622    Adult•kidney•
                                                                                       P01629 Mouse•whole•embryo,•
         Mouse•tissue•I•x-NubG
                                                                                              16d
         P01603    Adult•heart•               P01629 Normalized•whole•
                                                     embryo,17d
         P01616    Adult•spleen•

  44
                            Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter vectors   1.3.3




DUALhunter vectors
›› Vectors for the DUALhunter system


Applications
•• Discovery•of•novel•protein•interactions•by•screening•of•cDNA•libraries
•• • ompatible• with• nuclear• proteins,• transcription• factors,• acidic•
   C
   proteins•and•self-activating•proteins
•• Analysis•of•direct•protein•interactions
•• Domain•mapping•studies                                                    Ordering information

                                                                             Order•number   Product                                      Size
pDHB1 bait vector
                                                                             P03501         pDHB1•bait•vector                            5•µg
•• • tandard•bait•vector•for•the•DUALhunter•system.•The•protein•of•
   S                                                                         P03234         pPR3-N•prey•vector                           5•µg
   interest•is•fused•N-terminally•to•the•Ost4p•membrane•anchor•and•          P03243•        DUALhunter•control•plasmid•set               5•µg•each
   C-terminally•to•the•Cub-LexA-VP16•reporter•cassette.


pPR3-N prey vector
•• • rey•vector•for•membrane•proteins•with•cytosolic•N-terminus•and•
   P
   for•cytosolic•proteins.•


Control plasmid set
•• • •set•of•positive•and•negative•control•interactions•for•DUALhunter•
   A
   studies:•pTSU2-APP,•pNubG-Fe65,•pOST1-NubI.




                                                                                                                                                      45
1.3.4         Protein•interaction•discovery•|•Nuclear•proteins•and•transcription•factors•|•The•DUALhunter•system•|•DUALhunter cDNA libraries




              DUALhunter cDNA libraries
              ›› cDNA libraries for the DUALhunter system


        Applications                                                                   Order•    Product•                  Order• Product•
            •• • iscovery•of•novel•protein•interactions•by•DUALhunter•screening•
               D                                                                       number    Size:•200•µg              number Size:•200•µg
        •      of•cDNA•libraries                                                       Rat•cells•I•NubG-x
                                                                                       P02233    Neonatal•cardiomyocytes
                                                                                       Mouse•tissue•I•NubG-x
            Order•    Product•                  Order• Product•                        P02201    Adult•whole•brain•        P02234 Whole•embryo,•11d•
            number    Size:•200•µg              number Size:•200•µg                    P02207    Adult•heart               P02303 Normalized•brain•
            Human•tissue•I•NubG-x                                                      P02231    Adult•kidney•
            P02213    Embryonal•brain           P02226 Adult•kidney                    Mouse•tissue•I•x-NubG
            P02215    Adult•colon•              P02230 Adult•lung                      P02206    Adult•heart•              P02301 Normalized•whole•embryo,•17d
            P02220    Adult•liver•              P02302 Normalized•spleen•              P02224    Adult•spleen•
            P02221    Adult•brain•              P02304 Normalized•fetal•brain          Model•organisms•I•NubG-x
            Human•tissue•I•x-NubG                                                      P02210    A. thaliana               P02237 S. cerevisiae•
            P02208    Adult•kidney              P02227 Adult•brain                     P02218    C. elegans•adult•         P02222 C. elegans•eggs•
            Human•cell•lines•I•NubG-x                                                  P02223 D. melanogaster•whole•       P02229 D. melanogaster whole•
            P02205    Jurkat•T,•unst.           P02228 LNCaP                                  embryo•(16•hours)•                  adult
            P02212    HeLa                      P02232 Mammary•epithelial              Normalized•I•NubG-x
            Plant•I•NubG-x                                                             P02302 Human•spleen                 P02303 Mouse•brain•I•NubG-x
            P02236 Medicago•nodules             P02210 A. thaliana                     P02304 Human•fetal•brain
            Plant•I•x-NubG                                                             Normalized•I•x-NubG
            P02235    Medicago•nodules                                                 P02301 Mouse•whole•embryo


  46
                                                                                              Protein•interaction•discovery•|•Yeast reporter strains        1.4




Yeast reporter strains
›› Yeast reporter strains for DUALhybrid (yeast two-hybrid), DUALmembrane and DUALhunter systems


Applications                                                              NMY70

•• • eporter• strains• for• the• DUALhybrid,• DUALhunter• and••
   R                                                                      Triple• reporter• strain• for• high• stringency• yeast• two-hybrid• screening•
   DUALmembrane•systems                                                   using•LexA-based•two-hybrid•systems.•Compatible•with•Mate&Plate•
•• Reporter•strains•for•LexA-based•yeast•two-hybrid•systems               libraries•from•Clontech.
                                                                          MATa his3delta200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3
                                                                          ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4 ∆gal4

Reporter strains
                                                                          Ordering information
NMY51
                                                                           Order•number      Product                                  Size
Triple•reporter•strain•for•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•
and•DUALhunter•systems.•                                                   P04005            NMY51•yeast•reporter•strain              stab•culture
MATa his3delta200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3            P04006            NMY61•yeast•reporter•strain              stab•culture
ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4                                   P04008            NMY70•yeast•reporter•strain              stab•culture

NMY61
Mating•partner•for•reporter•strains•NMY51•and•NMY70.
MATalpha his3delta200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3
ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4




                                                                                                                                                           47
1.5.1      Protein•interaction•discovery•|•Yeast•kits•and•reagents•|•DS Yeast transformation kit




           DS Yeast transformation kit
           ›› The complete solution for high efficiency yeast transformation


        Applications                                                                     Kit contents
        •• • ontains•all•necessary•reagents•to•perform•small•scale•and•library•
           C                                                                             Contents                                           Size
           scale•high•efficiency•transformations•of•yeast
                                                                                         50%•PEG                                            50•ml
                                                                                         10•x•TE•Buffer                                     12•ml
                                                                                         1M•LiOAc•solution                                  12•ml
        Benefits
                                                                                         carrier•ssDNA•(2•mg/ml)                            2•ml
        •• • o•need•to•prepare•PEG,•single•stranded•DNA•or•LiOAc•-•just•use•
           N
           our•ready-made,•optimized•reagents•and•you’ll•never•have•a•problem•           Ordering information
           with•yeast•transformation•again!
        •• • ufficient• for• 5• library• scale• transformations• or• 80• small• scale•
           S                                                                             Order•number        Product                       Size
           transformations                                                               P01003              DS•Yeast•transformation•kit   1•kit


        Technology
        Many•people•think•that•yeast•transformation•is•as•simple•as•transformation•
        of•E.coli.•This•is•true•if•you•have•the•right•reagents•in•your•hands•since•
        the•quality•of•transformation•reagents•is•absolutely•crucial•to•achieve•
        high•transformation•efficiencies.
        Library•scale•transformations•in•particular•need•to•be•highly•efficient•in•
        order•to•reduce•the•rate•of•false•negative•interactors.•




  48
                                                                  Protein•interaction•discovery•|•Yeast•kits•and•reagents•|•Single-stranded carrier DNA               1.5.2




Single-stranded carrier DNA
›› Large-fragment, single-stranded carrier DNA of the highest quality, suitable for all types of yeast transformations


Applications                                                                   Stability testing of single-stranded carrier DNA at RT

 •• • igh•quality•single-stranded•carrier•DNA•especially•formulated•for•
    H




                                                                                    Percent DNA in size range 8-15 kb
                                                                                                                        100
    all•types•of•high•efficiency•yeast•transformations
                                                                                                                         80

Benefits                                                                                                                 60

 •• • nsheared•single-stranded•DNA:•significantly•larger•average•frag-
    U                                                                                                                    40
    ment•size•than•carrier•DNA•from•competitors
                                                                                                                         20
 •• Increased•transformation•efficiencies
 •• Specially•formulated•for•extended•stability•at•room•temperature                                                       0
                                                                                                                              2       4      6       8      10   12
 •• Up•to•50%•cheaper•than•competitors’•carrier•DNA
                                                                                                                                  Incubation time (weeks)
 •• Sufficient•for•125•small•scale•or•7•library•scale•transformations


Ordering information                                                           Accessory products
                                                                                •• DUALhybrid•kit• •page 12
  Order•number     Product                                       Size
                                                                                •• DS•Yeast•transformation•kit•                           page 48
  P06001           Single-stranded•carrier•DNA                   3•x•1•ml
                                                                                •• Lytic•Agent• page 52




                                                                                                                                                                      49
1.5.3      Protein•interaction•discovery•|•Yeast•kits•and•reagents•|•HTX beta-galactosidase assay kit




           HTX beta-galactosidase assay kit
           ›› Quantitative measurement of beta-galactosidase levels in cell extracts


        Applications
        •• Measurement•of•beta-galactosidase•activity
        •• • ompatible•with•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•
           C
           and•DUALhunter•systems



        Benefits                                                                          Grow cells in a         One-step substrate       Detection /
                                                                                          deep-well block         addition and lysis       Quantification
        •• • easure•beta-galactosidase•assays•for•96•reactions•in•less•than•
           M                                                                                                      (10 minutes)
           15•minutes
        •• Cheaper•and•more•reliable•than•ONPG•or•filter•assays
        •• Sufficient•reagents•for•4•x•96•assays                                       Kit contents

                                                                                       Component                                                Size
        Technology                                                                     One-step•lysis•and•assay•reagent•                        50•ml
        The•HTX•beta-galactosidase•assay•kit•determines•beta-galactosidase•
                                                                                       Dye•stock•solution•                                      250•µl
        activity•in•cell•lysates•from•yeast,•bacterial• or•mammalian• cells.•Both•
        semi-quantitative• and• quantitative• measurements• are• possible• in• a•
        high-throughput•fashion.•                                                      Ordering information

                                                                                       Order•number     Product                                   Size
                                                                                       P01002           HTX•beta-galactosidase•assay•kit          1•kit




  50
                                                       Protein•interaction•discovery•|•Yeast•kits•and•reagents•|•SpeedPrep yeast plasmid isolation kit   1.5.4




SpeedPrep yeast plasmid isolation kit
›› The complete solution for highly efficient plasmid extraction from yeast


Applications                                                                  Kit contents
•• • ptimized••extraction•of•plasmids•from•yeast•in•an•easy•spin•col-
   O                                                                          Contents                                                    Size
   umn•format•                                                                Solution•A                                                  10•ml
•• High•recovery•of•low•and•high•copy•number•plasmids
                                                                              Solution•B                                                  10•ml
                                                                              Solution•C                                                  20•ml
                                                                              Wash•Buffer•(Concentrate)                                   6•ml
Benefits
                                                                              Lytic•Agent                                                 lyophilized
•• • fficient•cell•wall•removal•prior•to•cell•lysis•enhances•plasmid•res-
   E                                                                          QuickEx•Spin•Columns                                        Pack•of•50
   cue•efficiency                                                             Collection•Tubes                                            Pack•of•50
•• Easy•to•use•spin•column•protocol•yields•highly•pure•plasmid
•• No•need•for•glass•beads,•phenol•or•DNA•precipitation
•• Sufficient•for•50•plasmid•purifications                                    Ordering information

                                                                              Order•number       Product                                   Size
Technology                                                                    P01014             SpeedPrep•yeast•plasmid•isolation•kit     1•kit
The•SpeedPrep•kit•is•a•straightforward•solution•for•extraction•of•high•
and•low•copy•number•plasmids•from•yeast.•High•efficiency•enzymatic•
removal•of•the•cell•wall•is•followed•by•cell•lysis•and•plasmid•is•purified•
over•a•spin•column•in•three•easy•steps.•The•purified•plasmid•DNA•is•
suitable•for•PCR•and•transformation•into•E. coli.




                                                                                                                                                         51
1.5.5      Protein•interaction•discovery•|•Yeast•kits•and•reagents•|•Lytic Agent




           Lytic Agent
           ›› Highly efficient plasmid extraction from yeast


        Applications                                                                Kit contents
        •• • ptimized••extraction•of•plasmids•from•yeast••in•an•easy•to•handle•
           O                                                                        Contents                                           Size
           96-well•format••                                                         Kit•for•96•Preps
        •• Compatible•with•all•commonly•used•plasmid•miniprep•kits
                                                                                    Lytic•Agent•Buffer                                 4•ml
                                                                                    Lytic•Agent•Powder                                 lyophilized
        Benefits                                                                    Kit•for•384•Preps
        •• No•mechanical•disruption•of•yeast•needed                                 Lytic•Agent•Buffer                                 16•ml
        •• • fficient•cell•wall•removal•prior•to•cell•lysis•enhances•plasmid•res-
           E                                                                        Lytic•Agent•Powder                                 lyophilized
           cue•efficiency                                                           Kit•for•3840•Preps
        •• Easy•to•combine•with•existing•plasmid•miniprep•kits
                                                                                    Lytic•Agent•Buffer                                 164•ml
                                                                                    Lytic•Agent•Powder•                                lyophilized
        Technology
        The•Lytic•Agent•is•an•enzymatic•mix•which•supports•plasmid•extraction•
        from•yeast•cells.•                                                          Ordering information
        Plasmid•DNA•purified•with•the•Lytic•Agent•can•be•used•in•downstream•
                                                                                    Order•number        Product                           Size
        applications,•such•as•PCR•amplification•or•transformation•of•E.coli.
                                                                                    P09101              Lytic•Agent•kit•(96•preps)        1•kit
                                                                                    P09102              Lytic•Agent•kit•(384•preps)       1•kit
                                                                                    P09103              Lytic•Agent•kit•(3840•preps)      1•kit




  52
                                                                             Protein•interaction•discovery•|•Yeast•kits•and•reagents•|•Antibodies   1.5.6




Antibodies
›› Detect your bait and prey proteins by Western blotting


Applications                                                            Anti LYTAG
 •• • ntibodies•for•the•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•
    A                                                                   Rabbit•polyclonal•antibody•against•the•LYTAG.•For•detection•of•fusion•
    and•DUALhunter•systems                                              proteins•in•the•DUALXpress•system.
 •• Antibodies•against•epitope•tags                                     Sufficient•for•10•Western•blot•reactions.


Anti LexA                                                               Anti ECS
Mouse•monoclonal•antibody•against•the•LexA•protein.•For•detection•of•   Rabbit•polyclonal•antibody•against•the•popular•ECS•(DDDDK)•tag*.•
baits•in•DUALhybrid•(yeast•two-hybrid),•DUALhunter•and•DUALmem-         Sufficient•for•10•Western•blot•reactions.•
brane•systems.
Sufficient•for•20•Western•blot•reactions.                               Ordering information

                                                                         Order•number     Product                                      Size
Anti HA
                                                                         P06004           Anti•LexA                                    1•vial
Mouse•monoclonal•antibody•against•the•HA•epitope•tag.•For•detection•
                                                                         P06005           Anti•HA                                      1•vial
of•preys•in•DUALhybrid•(yeast•two-hybrid),•DUALhunter•and•DUALmem-
                                                                         P06006           Anti•c-myc                                   1•vial
brane•systems.
                                                                         P06007           Anti•LYTAG                                   1•vial
Sufficient•for•10•Western•blot•reactions.
                                                                         P06008           Anti•ECS                                     1•vial

Anti c-myc
Mouse•monoclonal•antibody•against•the•c-myc•epitope•tag.                *•The•ECS•(DDDDK)•tag•is•identical•to•the•FLAG•tag.
Sufficient•for•20•Western•blot•reactions.


                                                                                                                                                    53
1.5.7      Protein•interaction•discovery•|•Yeast•kits•and•reagents•|•Media packages




           Media packages 1 & 2
           ›› Optimized growth media for all types of yeast two-hybrid applications


        Applications                                                                  To•complete•a•full•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•or•
                                                                                      DUALhunter•screen•including•analysis•of•prey•clones,•the•media•package•
        •• • omplete•media•packages•for•up•to•5•yeast•screens•(Media•pack-
           C
                                                                                      1•and•the•media•package•2•are•required.
           age•1)•and•analysis•of•up•to•100•preys•(Media•package•2)
        •• • ompatible•with•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•
           C
           and•DUALhunter•systems•                                                    Kit contents

                                                                                       Contents              Media•Pck.•1           Media•Pck.•2
        Benefits                                                                       YPAD•media            5•pouches              1•pouch
        •• No•need•for•time-consuming•dropout•medium•assembly                          SD-AHLW•media         21•pouches             11•pouches
        •• • eady-to-use•formulation•for•use•together•with•DUALhybrid•(yeast•
           R                                                                           Adenine               0.2•g                  0.2•g
           two-hybrid),•DUALmembrane•and•DUALhunter•kits                               L-histidine           0.2•g                  0.2•g
                                                                                       L-leucine             0.2•g                  0.2•g
        Technology
                                                                                       L-tryptophane         0.2•g                  0.2•g
        Media package 1                                                                3-aminotriazole       16.8•g                 4.4•g
        The•media•package•1•contains•all•growth•media•to•perform•up•to•five•
        DUALmembrane,•DUALhunter•or•DUALhybrid•(yeast•two-hybrid)•screens.•
                                                                                      Ordering information
        Sufficient• reagents• are•provided• for•all•steps•up•to•and•including• the•
        library•screening•and•clone•picking•stage.                                     Order•number      Product                                    Size
                                                                                       P09001            Media•package•1                            1•kit
        Media package 2                                                                P09002            Media•package•2                            1•kit
        The•media•package•2•contains•all•growth•media•to•perform•prey•analysis•
        of•up•to•100•preys•resulting•from•a•screen,•including•bait•dependency•
        and•confirmation•assays.

  54
                                                                                                                                Drug profiling services           2




Drug profiling services
›› A precise target fingerprint for drug candidates


Applications                                                                Technology
 •• Identify•direct•targets•for•small•molecule•drugs
 •• Elucidate•the•mechanism•of•action•for•drugs•under•development
 •• Become•aware•of•alternative•indications•for•drugs                           A




Benefits                                                                        B
Work in an intact cellular environment
 •• In situ•screening•increases•specificity•
 •• Weak•and/or•transient•interactions•are•detectable•(µM•range)
 •• • creening•of•normalized•universal•cDNA•libraries•ensures•exhaustive•
    S                                                                           C
    coverage•of•the•target•space
                                                                            Yeast•expressing•a•scaffold•protein•(DHFR)•fused•to•LexA•is•treated•with•a•
Lower your risks during drug development                                    drug•derivate•consisting•of•a•drug•of•interest•linked•to•a•methotrexate•anchor•
 •• Obtain•information•about•direct•targets•of•your•drug                    moiety•(A).•The•drug•derivate•binds•to•LexA-DHFR•via•its•methotrexate•part•
 •• Identify•the•mechanism•of•action•of•your•drug                           and•displays•the•drug•of•interest•as•a•“small•molecule•bait”•(B).•Subsequent•
                                                                            screening•against•a•cDNA•library•expressing•potential•targets•from•a•tissue•of•
Speed up your drug development                                              interest•as•fusions•to•the•activation•domain•of•Gal4•allows•the•rapid•isolation•
                                                                            of•drug-target•interactions.•
 •• Complete•profiling•of•your•drug•in•12•weeks
                                                                            Interaction•of•a•target•with•the•drug•of•interest•leads•to•assembly•of•a•hybrid•
                                                                            transcription•factor•from•the•LexA-DHFR•and•target-Gal4•fusion•proteins•via•
                                                                            the•small•molecule•derivate.•The•hybrid•transcription•factor•activates•a•reporter•
                                                                            gene,•whose•activity•is•measured•to•detect•the•drug-target•interaction•(C).
                                                                                                                                                                 55
2      Drug profiling services




     Custom screening procedure
     Step 1: Compound derivatization and toxicity test | 4 — 8 weeks
                                                                                        Drug of interest             Drug derivative (CID)
     •• You•provide•us•with•a•small•molecule
     •• • he•small•molecule•is•synthesized•as•a•derivative•linked•to•an•anchor•moiety
        T
     •• • he•anchor•moiety•serves•to•couple•the•derivatized•compound•to•the•
        T
                                                                                                                                             ()
                                                                                                                                              5




        bait•scaffold•and•a•flexible•linker•ensures•that•no•sterical•hindrance•
        occurs.

     Optional:•Synthesis•may•be•performed•by•customer
                                                                                                                          Drug          Linker       Anchor




     Step 2: cDNA library transformation | 2 — 4 weeks
                                                                                        Drug derivative (CID)                           Target tissue cDNA library
     •• • he•derivatized•compound•is•screened•against•a•cDNA•library•of•
        T
        interest•or•against•a•human•normalized•cDNA•library•covering•ap-
                                                                                                                ()
        prox.•95%•of•all•human•genes.•                                                                           5




     •• For•a•complete•list•of•cDNA•libraries•please•see• page 10




56
                                                                                     Drug profiling services        2




Step 3: Clone selection and analysis | 2 — 4 weeks
•• Selection•for•positive•interactions•on•selective•plates
•• Each•yeast•colony•represents•one•specific•interaction                     In situ growth selection
•• Identification•of•interacting•proteins•by•sequencing•and•BLAST•analysis




Step 4: Compound dependency test | 1 — 2 weeks
•• Compound-dependent•interactors•are•selectively•isolated

Deliverables                                                                                            specific
                                                                                                        target
•• Focused•list•of•highly•specific•targets•for•your•small•molecule•drug

Follow up assays
                                                                                                        noise
•• Competition•assays
•• In vitro binding•assays•(e.g.•pulldown•assays,•Biacore•studies)
•• Validation•of•interactions•in•human•cells

For more detail information, please visit our website:
www.dualsystems.com/Drugprofiling



                                                                                                                   57
3.1        cDNA library construction




           cDNA library construction
           ›› Construction of custom cDNA libraries for yeast-based screening systems and other applications


      Applications
                                                                                       A I First strand cDNA synthesis
      •• • DNA•libraries•for•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•
         c                                                                                       5' mRNA                                  AAAA 3'
         and•DUALhunter•systems                                                                                          3'-end adapter
      •• Custom•cDNA•library•construction•in•a•vector•of•your•choice                                                     TTTT
      •• Available•as•custom•service•and•as•kit                                                                                           AAAA
                                                                                                                                          TTTT
                                                                                                                         Easyclone RT
      Benefits
                                                                                                                                          AAAA
                                                                                                 CCCC                                     TTTT
      ••   High•representation•of•5’•ends
      ••   Guaranteed•coverage•of•tissue•complexity
      ••   Large•average•insert•sizes
      ••   Constructed•in•a•vector•of•your•choice                                      B I PlugOligo incorporation       PlugOligo       p
                                                                                                                                        T
      ••   Normalization•of•cDNA•available                                                                                    GGGG
                                                                                                          p                 IP solution
                                                                                                         T
      Construction process                                                                       GGGG
                                                                                                                                          AAAA
                                                                                                 CCCC                                     TTTT
      ••   Isolation•of•total•or•polyA+•RNA
      ••   Quality•control•on•Experion•chip
      ••   1st•strand•cDNA•generation                                                  C I ds cDNA preparation by PCR
      ••   Optional:•Normalization•of•cDNA,•confirmation•by•qPCR
                                                                                                                         PCR Primer M1
      ••   Size•fractionation
                                                                                                 GGGG                                     AAAA
      ••   2nd•strand•cDNA•generation•on•two•independent•size•pools
                                                                                                 CCCC                                     TTTT
      ••   Quality•control•on•Experion•chip                                                                     Ampliflied cDNA
      ••   Directional•ligation•into•appropriate•vector
      ••   Amplification•of•primary•library
 58
                                                                                                                            cDNA library construction         3.1




RNA quality control by Experion chip and in silico gel                        Specifications
electrophoresis                                                               ••   Special•5’•adaptor•annealing•procedure•preserves•full-length•cDNAs
The•quality•of•a•cDNA•library•depends•on•the•purity•and•preservation•         ••   Three•different•lengths•of•5’•adaptors•enclose•all•three•reading•frames
of•the•starting•material.•We•examine•the•purified•total•RNA•by•Experion•      ••   Average•insert•sizes•>•1.4•kb
chip•analysis•and•in silico•gel•electrophoresis.•The•28S•and•18S•RNA•         ••   High•complexities•(1•x•106•-•5•x•107•independent•clones)
are•clearly•visible•as•peaks•and•strong•bands,•respectively.                  ••   C
                                                                                   • loned• into• DUALhybrid• (yeast• two-hybrid),• DUALmembrane• or•
                                                                                   DUALhunter•library•vectors,•or•a•vector•of•your•choice
In•case•of•high•quality•total•RNA,•the•ratio•of•the•intensities•of•the•28S•
and•18S•bands•is•1.5-2.5•:•1.
       Fluorescence




                                                                 28S

                                                                 18S

                         28S    18S
                         Time




                                                                                                                                                             59
3.1.1      cDNA•library•construction•|•Normalized cDNA libraries




           Normalized cDNA libraries
           ›› Normalized cDNA library construction and normalized cDNA libraries


        Benefits                                                                      Low abundance transcripts
                                                                                      Low•abundance•transcripts•encode•many•of•the•key•signalling•proteins•
        •• Normalization•removes•abundant•transcripts
                                                                                      of•a•cell.•They•are•transcribed•at•very•low•levels•and•make•up•less•than•
        •• Decreased•rate•of•false•positives•in•yeast-based•screenings
                                                                                      1 %•of•the•total•mRNA•population.•Yet,•they•encode•key•components•
        •• •ncreased• chance• of• identifying• rare• interactors• in• yeast-• ased•
           I                                                                b
                                                                                      and•are•often•the•main•targets•of•protein•interaction•hunts.
           screenings


        A few abundant transcripts dominate RNA populations                                                     80
                                                                                                                70




                                                                                       Relative RNA abundance
        Typical•eukaryotic•mRNA•populations•consist•of•three•abundance•classes:
                                                                                                                60
        High abundance transcripts                                                                              50
        High•abundance•transcripts•include•mainly•housekeeping•genes,•such•                                     40
        as•GAPDH,•actins•and•components•of•the•transcriptional•and•transla-
                                                                                                                30
        tional•machinery.•They•are•expressed•at•very•high•levels•-•housekeeping•
                                                                                                                20
        genes•make•up•80-90%•of•all•mRNAs•in•a•cell.•They•represent•the•most•
        frequent•class•of•false•positive•interactions•in•yeast•two-hybrid•screens.                              10
                                                                                                                 0
                                                                                                                     High abundance Medium abundance   Low abundance
        Medium abundance transcripts                                                                                 transcripts    transcripts        transcripts
        These•transcripts•vary•depending•on•the•cell•type•and•make•up•10-20%•
        of•all•mRNAs.•                                                                                                 Native sample      Normalized sample




  60
                                                                                            cDNA•library•construction•|•Normalized cDNA libraries     3.1.1




Normalization process                                                       The•parameters•of•the•screens•and•the•resulting•interactors•are•shown•
                                                                            in•the•following•table.
A•double•stranded•cDNA•sample•is•normalized•based•on•differential•
re-association•kinetics•after•denaturation,•since•abundant•transcripts•
                                                                             Parameter                      Non-normalized•screen Normalized•screen
re-associate•faster•than•rare•ones.•Transcripts•are•denatured•and•then•
re-annealed• under• carefully• controlled• conditions.• The• reannealing•    Bait                           human•p53             human•p53
process•is•stopped•after•a•short•time,•when•the•majority•of•abundant•        Library                        human•colon           human•universal•
transcripts•have•reformed•into•double•stranded•cDNA,•whereas•rare•                                                                normalized
transcripts•are•still•single•stranded.                                       Screens•carried•out            3                     1
Subsequently,• double• stranded• cDNA• is• removed• by• digestion,• the•     Number•of•clones•screened      43•million            3.5•million
remaining• transcripts• are• denatured• again• and• annealed• to• double•    Number•of•interactors          436                   43
stranded•cDNA.•The•resulting•cDNA•population•is•normalized,•i.e.•the•        High•stringency•interactions   0                     12
majority•of•abundant•transcripts•has•been•removed.•
                                                                             Known•interactors              1                     3
Finally,•the•normalized•cDNA•sample•is•size•selected•and•ligated•into•
the•appropriate•vector.
                                                                            Normalization technology is available as custom screening serv-
                                                                            ice and as a kit.
Yeast two-hybrid screen using a normalized human                            •• • lease•see• •page 62•for•normalized•cDNA•library•construction•
                                                                               P
universal cDNA library                                                         services
                                                                            •• Please•see• •page 63•for•cDNA•library•construction•kits
Two•sets•of•yeast•two-hybrid•screens•were•carried•out•using•an•N-
terminally•truncated•form•of•human•p53•as•a•bait.•For•screening,•we•
used•two•libraries:
 •• A•standard•(non-normalized)•human•colon•library
 •• A•normalized•human•universal•library

                                                                                                                                                      61
3.1.2        cDNA•library•construction•| cDNA library construction services




             cDNA library construction services
             ›› Construction of custom cDNA libraries for yeast-based screening systems and other applications


        Applications                                                                 ••   Size•fractionation
                                                                                     ••   2nd•strand•cDNA•generation•on•two•independent•size•pools
        •• • DNA•libraries•for•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•
           c
                                                                                     ••   Quality•control•on•Experion•chip
           and•DUALhunter•systems
                                                                                     ••   Directional•ligation•into•appropriate•vector
        •• Custom•cDNA•library•construction•in•a•vector•of•your•choice
                                                                                     ••   Amplification•of•primary•library

        Benefits                                                                    Specifications
        ••   High•representation•of•5’•ends
                                                                                     •• Average•insert•sizes•>•1.4•kb
        ••   Guaranteed•coverage•of•tissue•complexity
                                                                                     •• High•complexities•(1•x•106•—•5•x•107•independent•clones)•
        ••   High•average•insert•sizes
                                                                                     •• • loned•into•DUALhybrid•(yeast•two-hybrid),•DUALmembrane,•or•
                                                                                        C
        ••   Normalization•of•cDNA•available
                                                                                        DUALhunter•library•vectors,•or•a•vector•of•your•choice
                                                                                     •• Average•turnaround•time•2•—•3•months
        Requirements                                                                 •• You•receive•primary•glycerol•stocks•and•purified•plasmid•DNA

        •• 5•µg•total•or•polyA+•RNA
        •• Sufficient•tissue•for•preparation•of•5•µg•polyA+•RNA                     Ordering information
        •• 5•aliquots•with•5•x•107•cells                                              Order•number       Service
                                                                                      S03002             Custom•standard•cDNA•library•construction
        Construction process                                                          S03012             Custom•normalized•cDNA•library•construction
        ••   Isolation•of•total•or•polyA+•RNA                                         S03000             Total•RNA•isolation•from•cells•or•tissue
        ••   Quality•control•on•Experion•chip                                         S03001             Poly(A)+•RNA•isolation•from•total•RNA
        ••   •1st•strand•cDNA•generation                                              S03004             Vector•modification•for•cDNA•library•construction
        ••   Optional: Normalization of cDNA, confirmation by qPCR

  62
                                                                               cDNA•library•construction•|•EasyClone cDNA library construction kits       3.1.3




EasyClone cDNA library construction kits
›› Construction of directional cDNA libraries from total or polyA+ RNA


Applications                                                                                            Reverse•transcription
 •• Directional•cDNA•library•construction
 •• • ompatible•with•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•
    C                                                                                                  ds•cDNA•amplification
    and•DUALhunter•systems
                                                                                                         Size•fractionation
Benefits
                                                                                       Ligation•into•appropriate•vector•&•library•amplification
 •• Fast•and•easy•synthesis•of•highly•complex,•representational•cDNAs
 •• High•representation•of•full•length•transcripts
 •• • irectional•cloning•of•cDNAs•into•DUALhybrid•(yeast•two-hybrid),• EasyClone normalized cDNA library construction
    D
    DUALmembrane•and•DUALhybrid•vectors                                package
 •• Only•small•starting•amounts•of•RNA•required                        •• Quick•and•easy•construction•of•normalized•cDNA•libraries
 •• Sufficient•reagents•to•construct•10•libraries                      •• Only•small•amounts•of•starting•RNA•required
                                                                       •• Directional•cloning
EasyClone cDNA library construction kit
                                                                              Normalization• is• the• process• of• removing• abundant• RNAs• from• the•
EasyClone•cDNA•synthesis•is•based•on•a•novel•technology•to•create•full•
                                                                              starting• material.• Normalized• cDNA• libraries• contain• approximately•
length•enriched•double•stranded•cDNA•from•total•or•polyA+•RNA•samples.•
                                                                              equal•amounts•of•all•RNAs•and•therefore•increase•the•occurrence•of•rare•
No•extensive•know-how•in•library•construction•is•needed•-•the•kit•sup-
                                                                              proteins.•This•significantly•increases•the•quality•of•DUALhybrid•(yeast•
plies•all•reagents•for•library•construction•and•the•detailed•manual•covers•
                                                                              two-hybrid),•DUALmembrane•and•DUALhunter•screens.
all•steps•of•library•construction.




                                                                                                                                                          63
3.1.3      cDNA•library•construction•|•EasyClone cDNA library construction kits




                                                                                  EasyClone PCR kit
          120
                                                                                  The•EasyClone•PCR•kits•contains•an•optimized•mixture•of•proofread-
          100
                                                                                  ing•polymerases•to•ensure•highly•accurate,•representational•PCR’s•and•
           80                                                                     preferred•amplification•of•large•fragments•from•complex•cDNA•mixtures.•
           60
           40
                                                                                                                 15•different•templates,•ranging•in•size•from•
                                                                                   3000bp                        0.6•kb•to•3•kb,•were•amplified•in•one•mul-
           20                                                                      2000bp
                                                                                                                 tiplex•reaction•using•the•EasyClone•PCR•
            0                                                                      1500bp
                                                                                                                 kit.•5•µl•of•the•reaction•was•loaded•onto•a•
                       non-normalized                  normalized
                                                                                   1000bp                        1%•agarose•gel,•flanked•by•size•markers.
                  Actin       hGAPDH                                                800bp
                                                                                    600bp
        qPCR of normalized and non-normalized human spleen cDNA. For actin, a
        decrease of 75.8% was reached. For hGAPDH, the decrease was 60.6%.
        The amount of these two high abundant cDNAs was lowered, increasing
        the quality of the cDNA library.                                          Ordering information

                                                                                   Order•number Product                                       Size
                                                                                   P01010         EasyClone•cDNA•library•construction•kit     20•reactions
                                                                                   P01011         EasyClone•normalized•cDNA•library•          20•reactions
                                                                                                  construction•package
                                                                                   P01012         EasyClone•PCR•kit                           100•reactions



  64
                                                        Protein•interaction•and•activity•assays•|•In vitro•binding•assays•|•Multiwell protein binding plates    4.1.1




Multiwell protein binding plates
›› Quick and simple detection of direct protein-protein interactions in vitro


Applications                                                                     The•binding•reactions•in•the•96-well•plate•format•may•be•used•for•ELISA•
                                                                                 and•studies•of•protein-protein,•protein-peptide,•protein-DNA•or•protein-
 •• • imple•method•to•detect•protein-protein,•protein-DNA•and•protein-
    S
                                                                                 RNA•interactions.
    RNA•interactions•in•a•96-well•format

                                                                                 Ordering information
Benefits                                                                          Order•       Product                                                Size
                                                                                  number
 •• Any•type•of•protein•can•be•used
                                                                                  P05101       96-well•anionic•protein-binding•plate•(black)          1•plate
 •• Generate•binding•curves•and•determine•the•Kd
 •• Easy•detection•by•fluorescence•or•light•absorption                            P05102       96-well•anionic•protein-binding•plate•(transparent)    1•plate
                                                                                  P05103       96-well•cationic•protein-binding•plate•(black)         1•plate

Technology                                                                        P05104       96-well•cationic•protein-binding•plate•(transparent)   1•plate

96-well•protein-binding•plates•can•be•used•to•study•specific•protein-protein,•
protein-DNA•or•protein-RNA•interactions.•The•anionic•protein-binding•
plates•bind•negatively•charged•proteins.•The•cationic•protein-binding•
plates•bind•positively•charged•proteins.•Since•the•bound-proteins•often•
maintain•their•native•confirmation•and•their•catalytic•or•binding•activities,•
the•bound•protein•can•be•recognized•by•a•second•molecule•that•spe-
cifically•interacts•with•the•bound•protein•where•the•native•conformation•
is•required.•The•second•molecule•can•be•detected•by•biochemical•or•
biophysical•methods.•Black•plates•provide•a•minimum•noise•level•for•
fluorescence•detection.•Transparent•plates•allow•detection•of•the•bound•
molecules•based•on•light•absorbance.•

                                                                                                                                                                65
4.2.1      Protein•interaction•and•activity•assays•|•Immunoprecipitation•assays•|•FleX-IP immunoprecipitation kit




           FleX-IP immunoprecipitation kit
           ›› Detection of protein interactions by co-immunoprecipitation


        Applications                                                                   The•FleX-IP•kit•is•optimally•suited•to•confirm•protein-protein•interac-
                                                                                       tions•found•in•DUALhybrid•(yeast•two-hybrid),•DUALmembrane•and•
        •• Immunoprecipitation•of•proteins•from•cells•and•cell-free•reactions
                                                                                       DUALhunter•screens•by•co-immunoprecipitation.
        •• Co-immunoprecipitation•experiments•to•validate•protein•interactions
        •• •deal• for• follow-up• studies• after• DUALhybrid• (yeast• two-hybrid),•
           I
           DUALmembrane•and•DUALhunter•screens                                         Kit contents

                                                                                        Contents                                       Size
        Benefits                                                                        Protein•A/G•agarose                            800•µl•(200•µl•resin)

        •• Complete•solution•for•easy•immunoprecipitation•experiments                   Control•mouse•IgG                              20•µl•(1•mg/ml)
        •• Optimized•lysis•and•wash•buffers                                             Lysis/Wash•buffer                              50•ml
        •• • pin•columns•for•easy•handling•and•improved•recovery•of•immune•
           S                                                                            NaCl•stock•solution•(5M)                       1•ml
           complexes                                                                    SDS-PAGE•buffer                                2•x•1•ml
        •• Compatible•with•mouse,•rat,•rabbit,•goat•and•human•antibodies                Spin•columns                                   40•columns
        •• Sufficient•for•40•IP•reactions
                                                                                        Collection•tubes                               40•tubes

        Technology
                                                                                       Ordering information
        The• FleX-IP• immunoprecipitation• kit• enables• highly• efficient• antigen•
                                                                                        Order•number        Product                                   Size
        immunoprecipitations•using•less•than•10•µg•of•antibody.•The•specific•
        antibody•is•first•added•to•the•sample•to•form•an•immune•complex,•which•         P01015              FleX-IP•immunoprecipitation•kit           1•kit
        is•then•added•to•the•Protein•A/G•agarose.•The•complex•is•washed•to•
        remove•unbound•material,•and•the•bound•immune•complex•is•eluted•for•
        downstream•applications•like•Western•blotting.•


  66
                                                          Protein•interaction•and•activity•assays•|•Protein•activity•assays•|•Universal kinase assay kit•   4.3.1




Universal kinase assay kit
›› Straightforward method for the measurement of enzymatic kinase activities


Applications                                                                   Kit contents
 •• • easure•the•activities•of•purified•kinases•producing•ADP•at•a•level•
    M                                                                          Contents                                                        Size
    of•5•µM                                                                    20•x•MUK•Buffer                                                 500•µl
                                                                               100•x•MUK•Reagent•A                                             30•µl•
                                                                               100•x•MUK•Reagent•B                                             30•µl•
Benefits                                                                       10•x•Fluorescence•dye                                           300•µl
 •• Any•type•of•kinase•can•be•tested                                           ATP•(10•mM)                                                     30•µl
 •• Only•small•amounts•of•purified•kinase•needed
 •• • he•kit•reagents•are•sufficient•for•100•assays•of•purified•kinases•
    T
                                                                               Ordering information
    using•a•standard•black•or•white,•96-well•or•384-well•plate
                                                                               Order•number      Product                                       Size
Technology                                                                     P05301            Universal•kinase•assay•kit                    1•kit

The• Universal• kinase• assay• kit• is• based• on• measurement• of• ADP•
generated•from•the•kinase•reaction.•It•measures•the•activities•of•puri-
fied•kinases•producing•ADP•at•a•level•of•5•µM•or•higher.•The•assay•is•
fluorescence-based•and•can•be•carried•out•using•regular•black•or•white•
96-well•or•384-well•plates•or•micro-cuvettes.•




                                                                                                                                                            67
4.3.2      Protein•interaction•and•activity•assays•|•Protein•activity•assays•|•PARP-1•assay kit




           PARP-1 assay kit
           ›› Fluorescence-based measurement of PARP-1 activity


        Applications                                                                   Kit contents
        •• • nzymatic•assay•to•determine•the•activity•of•purified•PARP-1•by•
           E                                                                             Contents                                    Size
           measurement•of•fluorescently•labeled•PAR•(poly•ADP-ribose)                    10•x•Assay•Buffer                           800•µl
                                                                                         10•x•DNA•template                           50•µl
        Benefits                                                                         NAD+•(10•mM)                                30•µl
        •• Only•small•amounts•of•purified•PARP-1•needed                                  100•x•Fluorescence•dye                      200•µl
        •• • he•kit•includes•reagents•for•100•assays•of•human•PARP-1•in•a•
           T
           96-well•plate•format
                                                                                       Ordering information
        Technology                                                                       Order•number             Product            Size

        Poly•(ADP-ribose)•polymerase-1•(PARP-1)•responds•to•DNA•damage•                  P05302                   PARP-1•assay•kit   1•kit
        and•synthesizes•poly•ADP-ribose•(PAR)•that•is•transferred•to•itself•and•
        a•variety•of•acceptor•proteins.•Excessive•activation•of•PARP-1•not•only•
        depletes•NAD+•but•also•generates•toxic•PAR,•resulting•in•cell•death.••The•
        human•PARP-1•assay•kit•is•based•on•measurement•of•PAR•(poly•ADP-
        ribose)•binding•to•a•fluorescent•dye,•which•results•in•a•fluorescent•signal.




  68
                                                                                           Protein•expression•| Expression•in•E. coli•|•DUALXpress system              5.1.1




    DUALXpress system
    ›› Expression of toxic and insoluble proteins in E. coli


Applications                                                                      The•LYTAG•enhances•solubility•of•the•fusion•protein•and•allows•recovery•
                                                                                  of• at• least• some• protein• from• the• soluble• fraction• in• the• majority• of•
    •• Expression•of•toxic•proteins•in•E. coli
                                                                                  cases.•One-step•purification•using•the•highly•selective•dualTRAP•resin•
    •• Expression•of•insoluble•proteins•in•E. coli
                                                                                  allows•elimination•of•most•contaminants,•resulting•in•highly•pure•protein.•
                                                                                  The•LYTAG•can•be•removed•by•enterokinase•cleavage.
Benefits
    ••   Highly•regulated•system•allows•expression•of•toxic•proteins
    ••   LYTAG•enhances•solubility•of•fusion•proteins
    ••   One-step•purification•of•recombinant•protein•to•>•95%•purity
    ••   LYTAG•removable•by•enterokinase•cleavage
    ••   N-•and•C-terminal•fusion•to•LYTAG•possible
•                                                                                   Load extract       Wash column to          Transfer column      Elute pure
                                                                                    onto column        remove impurities       to fresh tube        protein
Technology
The•DUALXpress•system•uses•the•CASCADE™•technology•to•achieve•
tight•regulation•and•very•high•expression•levels.
           β-Galacosidase Activity




                                     120.000                          uninduced
                                     100.000                          induced
                                      80.000
                   (units)




                                      60.000
                                      40.000
                                      20.000
                                           0
                                                T7       DUALXpress
                                               Expression systems
                                                                                                                                                                       69
5.1.1    Protein•expression•| Expression•in•E. coli•|•DUALXpress system




        DUALXpress starter kit                                                 Ordering information

        Kit contents                                                           Order•number   Product                                 Size
                                                                               P01008         DUALXpress•starter•kit                  1•kit
         Component                                              Size
                                                                               P03601         pALEX2a•expression•vector               5•µg
         pALEX2a•expression•vector•(N-terminal•fusion)          5•µg
                                                                               P03602         pALEX2b•expression•vector               5•µg
         pALEX2-CT•expression•vector•(C-terminal•fusion)        5•µg
                                                                               P03603         pALEX2c•expression•vector               5•µg
         REG-12•E.coli expression•strain                        stab•culture
                                                                               P03604         pALEX2a,b,c•vector•set                  5•µg•each
         INDUCER•solution•(1M)                                  500•µl
                                                                               P03605         pALEX2-CT•expression•vector             5•µg
         PROLYSE•extraction•solution                            12•ml
                                                                               P04101         REG-12•E.coli expression•strain         stab•culture
         dualTRAP•spin•columns                                  10•pcs
                                                                               P06101         PROLYSE•extraction•solution             25•ml
         ELUTION•buffer•(3M)                                    500•µl
                                                                               P06102         PROLYSE•extraction•solution             100•ml
         Anti•LYTAG•polyclonal•antibody                         100•µl
                                                                               P06103         dualTRAP•affinity•resin                 30•ml
         WASH•buffer                                            40•ml
                                                                               P06104         dualTRAP•affinity•resin                 250•ml
                                                                               P06105         dualTRAP•spin•columns                   10•pcs
        DUALXpress reagents
                                                                               P06106         dualTRAP•spin•columns                   25•pcs
        All•DUALXpress•reagents•are•also•available•separately,•including•      P06107         dualTRAP•spin•columns                   100•pcs
        dualTRAP•affinity•resin•and•dualTRAP•multiwell•affinity•plates.
                                                                               P06108         dualTRAP•multiwell•affinity•plate       1•pc
                                                                               P06109         dualTRAP•multiwell•affinity•plates      5•pcs
                                                                               P06110         INDUCER•solution                        25•ml
                                                                               P06111         ELUTION•buffer                          20•ml
                                                                               P06007         Rabbit•polyclonal•anti•LYTAG•antibody   100•µl

  70
                                                                                      Protein•expression•| Expression•in•E. coli•|•QuickLyse™ system      5.1.2




QuickLyse™ system
›› Rapid autolysis of bacterial strains for recombinant protein purification


Applications
                                                                                     Clone•cDNA•encoding•protein•to•be•expressed•into•pEAS-1a
 •• Expression•of•recombinant•proteins•in•E. coli
 •• • ne-step•cell•lysis•and•release•of•protein•into•supernatant
    O                                                                            Transform•into•appropriate•expression•host•&•induce•protein•expression
 •• Compatible•with•all•purification•tags
                                                                                          Induce•cell•lysis•by•incubation•at•38°•C•for•2•hours

Benefits                                                                                  Extract•and•purify•protein•from•culture•supernatant
 •• Recombinant•proteins•are•purified•directly•from•the•culture•supernatant
 •• No•need•for•lysozyme•treatment•or•sonication
                                                                               References
 •• • entle•extraction•conditions•ensure•integrity•and•activity•of•the•re-
    G
    combinant•protein                                                          •• Xu•et•al.•Biotechniques•41,•19-322•(2006)
 •• Suitable•for•high-throughput•protein•expression                            •• Kloos•et•al.•J. Bacteriol.•176,•7352-7361•(1994)


Technology                                                                     Ordering information
The•QuickLyse™•system•provides•rapid,•temperature-controlled•lysis•of•         Order•number       Product                              Size
bacteria•expressing•your•recombinant•protein•of•interest.•The•protein•of•      P08001             pEAS-1a•expression•vector            5•µg
interest•is•expressed•from•the•vector•pEAS-1a,•followed•by•a•temperature•
shift•to•induce•autolysis.•After•lysis,•the•recombinant•protein•is•purified•
from•the•culture•supernatant.•The•system•is•compatible•with•most•
E. coli•strains,•such•as•BL21•or•XL1-Blue.




                                                                                                                                                          71
5.1.3      Protein•expression•| Expression•in•E. coli•|•Expression vectors




           Expression vectors for Escherichia coli
           ›› High-level protein expression in E. coli


        Applications                                                                 Increased protein production with alternative produc-
        •• Tightly•regulated•expression•of•recombinant•proteins••in E. coli          tion organisms
        •• Fermentation•studies                                                      Numerous•bacteria•can•be•used•for•protein•production•with•the•VB•vec-
        •• Antibiotic-free•cultivation•using•parDE•operon                            tors,•including•E. coli,•Pseudomonas spp.•and•Caulobacter crescentus,•
                                                                                     but•also•many•novel•organisms•that•are•promising•for•protein•production.•
                                                                                     Our•expression•vectors•can•be•used•to•easily•screen•for•the•perfect•
        Benefits                                                                     organism•for•production•of•any•protein.

        •• • ltra-tight•regulation•and•high•levels•of•expression•due•to•Pm/xylS•
           U
           regulon                                                                   Ordering / vector information
        •• • ompatible•with•most•expression•vectors,•including•pET,•pUC•and•
           C
                                                                                      Order•    Product• Promoter    Origin   Resist-       Resist-       Size
           pBAD•series
                                                                                      number    name                          ance•1        ance•2
        •• Wide•choice•of•antibiotic•resistance•markers
                                                                                      P03701    pVB1      Pm/xylS    RK2      Ampicillin    -             5•µg
        •• • K2•origin•allows•vector•propagation•in•more•than•60•gram•nega-
           R
           tive•species                                                               P03702    pVB2      Pm/xylS    RK2      Ampicillin    -             5•µg
                                                                                      P03703    pVB3      Pm/xylS    RK2      Ampicillin    -             5•µg
                                                                                      P03704    pVB4      Pm/xylS    RK2      Tetracyclin   -             5•µg
        pVB expression vectors
                                                                                      P03705    pVB5      Pm/xylS    RK2      Kanamycin     -             5•µg
        The•VB•expression•technology•comprises•the•most•versatile•and•sophis-
                                                                                      P03706    pVB6      -          RK2      Ampicillin    Chloram-      5•µg
        ticated•system•available•for•the•production•of•recombinant•proteins.•The•                                                           phenicol
        system•combines•two•unique•properties:•The•highly•customizable•Pm/
                                                                                      P03707    pVB7      -          RK2      Ampicillin    Tetracyclin   5•µg
        xylS•expression•system•and•a•broad-host-range•RK2•minimal•replicon.•
                                                                                      P03708    pVB8      -          RK2      Ampicillin    Kanamycin     5•µg
        The•VB•system•is•ideal•for•high-level,•low-cost,•controllable•recombinant•
        protein•production•in E. coli•and•other•gram•negative•bacteria.               P03709    pVB9      -          RK2      Ampicillin    parDE

  72
                                                                            Protein•expression•| Expression•in•yeast•|•KickStart expression system        5.2.1




KickStart expression system
›› Saccharomyces cerevisiae expression system


Applications                                                                 Technology
 •• Expression•of•recombinant•proteins•in•S. cerevisiae                      The• KickStart• expression• system• combines• two• crucial• features• for•
 •• Expression•of•membrane•proteins•and•secreted•proteins                    protein•expression:

                                                                             •• •nducible•ADH2•promoter:•The•ADH2•promoter•is•induced•when•
                                                                                I
Benefits                                                                        glucose• becomes• depleted• in• the• medium.• During• early• growth•
                                                                                in•YPD•medium•glucose•levels•are•high•and•the•ADH2•promoter•
 ••   Protein•expression•in•rich•medium•(YPD)                                   is•inactive•-•thereby•preventing•negative•effects•on•yeast•growth.•
 ••   Self-inducing•system,•no•addition•of•inducer•required                     Once•the•cells•reach•the•early•stationary•phase•glucose•is•depleted•
 ••   Protease•deficient•strain•for•higher•protein•yields                       from•the•medium•and•the•ADH2•promoter•is•induced,•producing•
 ••   One-step•purification•using•the•popular•Strep•and•ECS•(DDDDK)•tags*       high•amounts•of•protein.

                                                                             •• •• anMX•cassette:•KickStart•vectors•feature•a•KanMX•cassette,•which•
                                                                                 K
                                                                                 allows•selection•with•G418•in•rich•medium.•The•use•of•rich•medium•
                                                                                 such•as•YPD•significantly•enhances•cell•densities•and•protein•yields.

                                                                             •• Auto-inducible•ADH2•promoter:
                                                                                                    The•ADH2•promoter•is•repressed•by•glucose.•
                                                                                                     When• the• cells• reach• the• early• stationary•
                                                                                                     phase,•glucose•is•depleted•from•the•medium•
                                                                                                     (red• arrow)• and• the• ADH2• promoter• is• in-
                                                                                                     duced,•producing•high•amounts•of•protein.

                                                                             *The•ECS•(DDDDK)•tag•is•identical•to•the•FLAG•tag
                                                                                                                                                          73
5.2.1     Protein•expression•| Expression•in•yeast•|•KickStart expression system




        KickStart protein expression kit                                                Ordering information
        The•KickStart•protein•expression•kit•contains•expression•vectors,•strain•       Order•number   Product                                  Size
        and•purification•reagents•sufficient•for•at•least•10•purifications.•All•puri-   P01006         KickStart•protein•expression•kit         1•kit
        fication•reagents•can•also•be•purchased•separately.
                                                                                        P03305         pKS1•tagless•expression•vector           5•µg
                                                                                        P03306         pKS1-ST•Strep-tag•expression•vector      5•µg
        Kit contents                                                                    P03307         pKS1-ST-Gal•control•vector               5•µg
         Contents                                                    Size               P03308         pKS2-ST•Strep-tag•secreted•expression•   5•µg
                                                                                                       vector
         pKS1•tagless•expression•vector                              5•µg
                                                                                        P03309         pKS3-FL•ECS-tag•expression•vector        5•µg
         pKS1-ST•Strep-tag•expression•vector                         5•µg
                                                                                        P03310         pKS4-FL•ECS-tag•secretion•vector         5•µg
         pKS-ST-Gal•control•vector                                   5•µg
                                                                                        P04003         DSY-5•yeast•expression•strain            stab•
         DSY-5•expression•strain                                     stab•culture
                                                                                                                                                culture
         XT•extraction•buffer•                                       50•ml
                                                                                        P06003         XT•extraction•buffer/•glass•beads        300•m l/•
         Glass•beads•                                                20•ml                                                                      2•x•50•ml
                                                                                        P06201         ECS•affinity•resin                       0.2•ml
        KickStart reagents                                                              P06008         Anti•ECS•antibody,•for•10•immunoblot•    20•µl
                                                                                                       reactions
        All•KickStart•reagents•are•also•available•separately,•including•vectors,•
        strains•and•reagents.                                                           P01003         DS•Yeast•transformation•kit              1•kit
                                                                                        P06001         Single•stranded•carrier•DNA              3•x•1•ml




  74
                                                                              Protein•expression•| Expression•in•yeast•|•Yeast expression vectors      5.2.2




Yeast expression vectors
›› Intracellular and secreted expression in Saccharomyces cerevisiae


Applications                                                                 pTEF-MF
 •• Expression•of•recombinant•proteins•in•S. cerevisiae                      •• • east•expression•vector•for•secreted•proteins.•A•strong•TEF1•pro-
                                                                                Y
 •• Expression•of•membrane•proteins•and•secreted•proteins                       moter•drives•constitutive•expression•of•a•cDNA•fused•to•the•pre-pro•
 •• Inducible•expression                                                        leader•sequence•of•mating•factor•alpha•to•ensure•secretion•of•the•
                                                                                protein•product•into•the•medium.
Benefits
                                                                             pGAL-MF
 •• High•protein•yields•due•to•expression•in•rich•medium•(YPD)
 •• Expression•of•toxic•proteins•possible                                    •• • east•expression•vector•for•secreted•proteins.•A•regulated•GAL1-L•
                                                                                Y
                                                                                promoter•drives•inducible•expression•of•a•cDNA•fused•to•the•pre-
p427-TEF                                                                        pro•leader•sequence•of•mating•factor•alpha•to•ensure•secretion•of•
                                                                                the•protein•product•into•the•medium.•
 •• • igh• copy• yeast• expression• vector• carrying• the• aminoglycoside•
    H
    phosphotransferase•gene•for•selection•in•yeast•using•G418.•Inserts•
                                                                             Ordering information
    are•expressed•from•the•strong•TEF•promoter.
                                                                             Order•number     Product                                      Size
p417-CYC                                                                     P03301           p427-TEF                                     5•µg
                                                                             P03302           p417-CYC                                     5•µg
 •• • ow• copy• yeast• expression• vector• carrying• the• aminoglycoside•
    L
    phosphotransferase•gene•for•selection•in•yeast•using•G418.•Inserts•      P03303           pTEF-MF                                      5•µg
    are•expressed•from•the•weak•CYC1•promoter.                               P03304           pGAL-MF                                      5•µg




                                                                                                                                                       75
5.2.2    Protein•expression•| Expression•in•yeast•|•Yeast expression vectors




        pFunk expression vectors                                                Order•   Vector      Promoter   Origin    Marker   Size
        ••   General•purpose•yeast•expression•vectors                           number
        ••   Wide•choice•of•promoters•and•auxotrophic•markers                   P03331   p416CYC     CYC1       CEN/ARS   URA3     5•µg
        ••   Constitutive•and•inducible•expression                              P03332   p416GAL-S   GAL1-S     CEN/ARS   URA3     5•µg
        ••   Rich•multiple•cloning•site•for•easy•subcloning•of•cDNA•inserts     P03333   p416GAL-L   GAL1-L     CEN/ARS   URA3     5•µg
                                                                                P03334   p423CYC     CYC1       2micron   HIS3     5•µg
        Ordering information                                                    P03335   p423GAL-S   GAL1-S     2micron   HIS3     5•µg
                                                                                P03336   p423GAL-L   GAL1-L     2micron   HIS3     5•µg
        Order•           Vector         Promoter    Origin      Marker   Size
        number                                                                  P03337   p424CYC     CYC1       2micron   TRP1     5•µg

        P03320           p413CYC        CYC1        CEN/ARS     HIS3     5•µg   P03338   p424ADH     ADH1       2micron   TRP1     5•µg

        P03321           p413GAL-S      GAL1-S      CEN/ARS     HIS3     5•µg   P03339   p424TEF     TEF1       2micron   TRP1     5•µg

        P03322           p413GAL-L      GAL1-L      CEN/ARS     HIS3     5•µg   P03340   p425CYC     CYC1       2micron   LEU2     5•µg

        P03323           p414CYC        CYC1        CEN/ARS     TRP1     5•µg   P03341   p425ADH     ADH1       2micron   LEU2     5•µg

        P03324           p414TEF        TEF1        CEN/ARS     TRP1     5•µg   P03342   p425TEF     TEF1       2micron   LEU2     5•µg

        P03325           p414GAL-S      GAL1-S      CEN/ARS     TRP1     5•µg   P03343   p425GAL-S   GAL1-S     2micron   LEU2     5•µg

        P03326           p414GAL-L      GAL1-L      CEN/ARS     TRP1     5•µg   P03344   p425GAL-L   GAL1-L     2micron   LEU2     5•µg

        P03327           p415CYC        CYC1        CEN/ARS     LEU2     5•µg   P03345   p426CYC     CYC1       2micron   URA3     5•µg

        P03328           p415ADH        ADH1        CEN/ARS     LEU2     5•µg   P03346   p426TEF     TEF1       2micron   URA3     5•µg

        P03329           p415GAL-S      GAL1-S      CEN/ARS     LEU2     5•µg   P03347   p426GAL-S   GAL1-S     2micron   URA3     5•µg

        P03330           p415GAL-L      GAL1-L      CEN/ARS     LEU2     5•µg   P03348   p426GAL-L   GAL1-L     2micron   URA3     5•µg



  76
                                                                        Protein•expression•| Expression•in•yeast•|•Yeast expression vectors       5.2.2




Explanation of vector features
•• CYC1:•CYC1•promoter,•very•low•expression•level,•constitutive        •• TRP1:• TRP1• auxotrophic• marker,• allows• growth• of• yeast• trans-
                                                                          formed•with•TRP1-marked•plasmids•on•selective•medium•lacking•
•• ADH1:•ADH1•promoter,•strong•expression•level,•constitutive             tryptophan•(SD-W)

•• TEF1:•TEF1•promoter,•very•strong•expression•level,•constitutive     •• LEU2:• LEU2• auxotrophic• marker,• allows• growth• of• yeast• trans-
                                                                          formed•with•LEU2-marked•plasmids•on•selective•medium•lacking•
•• • AL1-S:•Short•variant•of•the•galactose-inducible•GAL1•promoter,•
   G                                                                      leucine•(SD-L)
   low•expression•level
                                                                       •• • RA3:• URA3• auxotrophic• marker,• allows• growth• of• yeast• trans-
                                                                          U
•• • AL1-L:•Long•variant•of•the•galactose-inducible•GAL1•promoter,•
   G                                                                      formed•with•URA3-marked•plasmids•on•selective•medium•lacking•
   intermediate•expression•level                                          uracil•(SD-U)

•• CEN/ARS:•low•copy•CEN/ARS•origin•(1-2•copies•per•cell)              •• HIS3:•HIS3•auxotrophic•marker,•allows•growth•of•yeast•transformed•
                                                                          with•HIS3-marked•plasmids•on•selective•medium•lacking•histidine•
•• 2micron:•high•copy•2•micron•origin•(20-50•copies•per•cell)             (SD-H)




                                                                                                                                                  77
5.2.3     Protein•expression•| Expression•in•yeast•|•Yeast expression strains




             Yeast expression strains
             ›› Protease deficient yeast expression strains


        Applications                                                                   Ordering information
        •• Expression•of•recombinant•proteins•in•yeast                                 Order•number   Product                         Size
        •• Intracellular•and•secreted•expression                                       P04003         DSY-5•yeast•expression•strain   stab•culture
        •• Protease•deficient•strains
                                                                                       P04007         DSY-6•yeast•expression•strain   stab•culture

        Expression strains
        DSY-5
        General•purpose•expression•strain.•Deletion•of•the•two•major•vacuolar•
        proteases• Pep4• and• Prb1• ensures• good• yield• and• little• non-specific•
        degradation•when•purifying•proteins•from•yeast.•
        MATalpha leu2 trp1 ura3-52 his3::GAL1-GAL4 pep4 prb1-1122

        DSY-6
        General• purpose• expression• strain.• Deletion• of• the• proteases• prb1,•
        pep4•and•prc1•results•in•minimal•protein•degradation•during•cell•lysis•
        and•protein•extraction.
        MATa leu2 trp1delta63 ura3-52 prb1-22 pep4-3 prc1-407




  78
                                                             Protein•expression•| Expression•in mammalian•cells•| Stable cell line generation services       5.3.1




Stable cell line generation services
›› Construction of stably transfected cell lines


Applications                                                                  Cell line generation process
 ••   Generation•of•a•stable•cell•line•expressing•your•protein•of•interest    Part I
 ••   Verification•of•expression•by•Western•blotting                          •• Construction•of•integration•vector•containing•your•gene•of•interest•
 ••   Complete•flexibility:•wide•choice•of•different•tags                     •• Transfection•into•appropriate•cell•line
 ••   Save•time•&•money:•6•weeks•turnaround•time•at•an•unbeatable•price
 ••   No•risks:•money•back•guarantee
                                                                                        Gene of
                                                                                        interest
                                                                                  HA
                                                                               Strep
Profit from our state-of-the-art integration technology
                                                                                          FRT
 •• Single•copy•integration•at•a•defined•site
                                                                                Bait construction    Stable integration    Selection     Expression test
 •• Physiological•expression•level
 •• Longterm•expression
                                                                              Different•tags•are•available,•tags•can•be•placed•either•at•the•N-•or•C-
                                                                              terminus.
                                                                              No•stable•lines•after•two•transfection•attempts:•only•fee•for•Part•I•is•due.

                                                                              Part II
                                                                              •• Selection•of•stable•clones
                                                                              •• Expansion
                                                                              •• Expression•test•by•Western•blotting

                                                                              No•expression•of•transgene•detectable:•only•50%•of•fee•for•Part•II•is•due.

                                                                                                                                                             79
5.3.1      Protein•expression•| Expression•in mammalian•cells•| Stable cell line generation services




                                                                                            Available cell lines
                                                                                            ••   HEK293•-•Human•embryonic•kidney•cells
                                                              Overexpressed protein         ••   INS-1•-•Beta•pancreatic•islet•cells
                                                              Endogenous protein            ••   CHO•-•Chinese•hamster•ovary
                                                                                            ••   • T3•-•Mouse•embryonic•fibroblast•cells•
                                                                                                 3

                                                              Overexpressed protein
                                                              Endogenous protein


                                                              Overexpressed protein
                                                              Endogenous protein




        Comparison of protein expression levels of overexpressed proteins with en-          Ordering information
        dogenous protein levels. HEK293 cell lines expressing tagged proteins were
                                                                                            Order•number     Service
        analysed by immunoblotting either prior to (uninduced) or after induction of bait
        protein expression (induced) using antibodies against the indicated proteins.       S07080           Stable•cell•line•generation•part•I
                                                                                            S07081           Stable•cell•line•generation•part•II
        Deliverables
        •• •sogenic•cell•pools•expressing•the•protein•of•interest•(5•vials•of•frozen•
           I                                                                                Related custom services
           stocks,•at•least•5•million•cells•per•vial)
                                                                                            •• Protein•interaction•screening•services• •see page 5
        •• Western•blot•demonstrating•protein•expression
                                                                                            •• cDNA•library•construction•services• •see page 62
        •• Cell•cultivation•protocol

  80
                                                              Protein•expression•| Expression•in mammalian•cells•| Mammalian expression vectors         5.3.2




Mammalian expression vectors
›› Protein expression in mammalian cells


Applications                                                                pMEX-HA / pMEX-FL
 •• Expression•of•recombinant•proteins•in•mammalian•cells                   Mammalian• expression• vectors• featuring• a• strong• CMV• promoter,• a•
 •• Transient•and•stable•expression                                         C-terminal•HA•or•ECS*•epitope•tag•for•detection•of•expressed•proteins•
 •• • ollow-up•co-immunoprecipitation•studies•after•DUALhybrid•(yeast•
    F                                                                       and•a•NeoR•cassette•for•stable•integration•of•the•expression•vector•into•
    two-hybrid),•DUALmembrane•and•DUALhunter•screens                        the•genome•of•the•host•cell.•pMEX-HA•and•pMEX-FL•are•suited•for•both•
                                                                            transient•overexpression•and•long-term,•stable•expression.


Benefits
 •• Strong•CMV•promoter•for•constitutive,•high-level•expression
 •• HA•and•ECS*•epitope•tags•for•immunoprecipitation/detection
                                                                            Ordering information
 •• Sfi•I•sites•for•convenient,•directional•cloning•of•full-length•cDNAs
                                                                             Order•number       Product                                 Size
pHA-MEX / pFL-MEX                                                            P03401             pHA-MEX                                 5•µg
                                                                             P03402             pMEX-HA                                 5•µg
Mammalian•expression•vectors•featuring•a•strong•CMV•promoter,•an•
N-terminal•HA•or•ECS*•epitope•tag•for•detection•of•expressed•proteins•       P03403             pFL-MEX                                 5•µg
and•a•NeoR•cassette•for•stable•integration•of•the•expression•vector•into•    P03404             pMEX-FL                                 5•µg
the•genome•of•the•host•cell.•pHA-MEX•and•pFL-MEX•are•suited•for•both•
transient•overexpression•and•long-term,•stable•expression.•                 *•The•ECS•(DDDDK)•epitope•tag•is•identical•to•the•FLAG•tag




                                                                                                                                                        81
6.1.1     Protein•extraction•|•Extraction•from•mammalian•cells•|•DUALXtract mammalian cell total protein extraction reagent




           DUALXtract mammalian cell total protein extraction reagent
           ›› Highly effective and convenient total protein extraction from mammalian cultured cells or tissue samples


        Applications                                                                     Ordering information
        •• Total•protein•extraction•from•mammalian•cells•or•tissues                      Order•number   Product                                    Size
                                                                                         P07113         DUALXtract•mammalian•cell•total•protein•   250•ml
                                                                                                        extraction•reagent
        Benefits
        •• One-step•extraction•of•complex•protein•samples
        •• No•sonication•and•no•freeze-thaw•cycles•required
        •• • entle• extraction• conditions• ensure• integrity• and• activity• of• the•
           G
           protein•sample
        •• • ufficient•for•125•extractions•from•100•μl•of•wet•cell•pellet•or•500•
           S
           extractions•of•100•mg•of•tissue


        Technology
        Proteins•extracted•using•this•reagent•are•non-denatured,•functionally•
        active•and•therefore•ideal•for•direct•use•in•many•common•downstream•
        applications,• including• 1D• and• 2D• electrophoresis,• Western• blotting,•
        electrophoretic•mobility•shift•assay•(EMSA),•immunoprecipitation,•affinity•
        purification,•enzymatic•activity•and•reporter•gene•assays.•Isolated•proteins•
        are•compatible•with•the•TOTAL•protein•assay•reagent•(see• •page 87)




  82
                                            Protein•extraction•|•Extraction•from•mammalian•cells•| DUALXtract total membrane protein extraction kit          6.1.2




DUALXtract total membrane protein extraction kit
›› Efficient isolation of membrane proteins from mammalian cells and tissue samples


Applications
 •• • apid•isolation•of•high•quality•membrane•and•cytoplasmic•protein•
    R
    fractions•from•mammalian•cells•and•tissues



Benefits
 •• No•cross-contamination•of•cytoplasmic•and•membrane•fractions
 •• Sufficient•for•50•extractions•from•5•million•cells•or•20-40•mg•of•tissue       Kit contents

                                                                                   Contents                                                       Size
Technology                                                                         Cell•Permeabilization•Buffer                                   100•ml
The•procedure•is•based•on•stepwise•differential•protein•solubilization.•           Membrane•Protein•Extraction•Buffer                             50•ml
Treatment•with•different•mild,•non-denaturing•detergent-based•buffers•             Cell•Wash•Solution                                             250•ml
selectively•separates•membrane•and•membrane-associated•proteins•
from•the•cytoplasmic•fraction.•
                                                                                   Ordering information
Cells• are• first• permeabilized• with• the• Cell• Permeabilization• Buffer• to•
release•the•cytoplasmic•protein•fraction.•The•cell•debris•is•then•treated•         Order•      Product                                               Size
with•the•Membrane•Protein•Extraction•Buffer•which•selectively•solubilizes•         number
the•majority•of•integral•and•membrane-associated•proteins•and•isolates•            P07115      DUALXtract•total•membrane•protein•extraction•kit      1•kit
them•as•a•separate•fraction.




                                                                                                                                                             83
6.1.3     Protein•extraction•|•Extraction•from•mammalian•cells•|•DUALXtract membrane protein optimization kits




             DUALXtract membrane protein optimization kits
             ›› Optimized extraction of integral membrane proteins and membrane-associated proteins


        Applications                                                                  Ordering information
        •• • uickly•identify•the•optimal•extraction•conditions•for•your•mem-
           Q                                                                          Order•number   Product                                             Size
           brane•protein•of•interest                                                  P07001         DUALXtract•buffer•set•(trial•size,•8•extractions)   1•kit
        •• • uitable•for•extraction•of•native,•functional•membrane•proteins•from•
           S
                                                                                      P07002         DUALXtract•buffer•set•(80•extractions)              1•kit
           mammalian,•fungal•and•bacterial•cells
                                                                                      P07101         DUALXtract•buffer•1                                 40•ml
                                                                                      P07102         DUALXtract•buffer•2                                 40•ml
        Benefits                                                                      P07103         DUALXtract•buffer•3                                 40•ml
        •• Quantitative•extraction•of•native•membrane•proteins                        P07104         DUALXtract•buffer•4                                 40•ml
        •• 12•different•extraction•conditions•for•optimization                        P07105         DUALXtract•buffer•5                                 40•ml
        •• • on-denaturating•detergents•and•synthetic•lipid•analogues•provide•
           N                                                                          P07106         DUALXtract•buffer•6                                 40•ml
           gentle•extraction•and•stabilization
                                                                                      P07107         DUALXtract•buffer•7                                 40•ml
                                                                                      P07108         DUALXtract•buffer•8                                 40•ml
        Step 1
                                                                                      P07109         DUALXtract•buffer•9                                 40•ml
        Use•the•DUALXtract•buffer•set•to•test•12•different•extraction•buffers•on•
                                                                                      P07110         DUALXtract•buffer•10                                40•ml
        your•membrane•protein•of•interest
                                                                                      P07111         DUALXtract•buffer•11                                40•ml
        Step 2                                                                        P07112         DUALXtract•buffer•12                                40•ml

        Once•you•have•identified•the•optimal•extraction•buffer,•use•this•buffer•to•
        perform•a•large•scale•extraction

        Step 3
        Continue•with•downstream•work•using•the•extracted•membrane•protein

  84
                                 Protein•extraction•|•Extraction•from•mammalian•cells•|•DUALXtract nuclear and cytoplasmic protein extraction kit            6.1.4




DUALXtract nuclear and cytoplasmic protein extraction kit
›› Rapid stepwise isolation of cytoplasmic and nuclear proteins from mammalian cultured cells and tissue


Applications
 •• • xtraction•of•cytoplasmic•proteins•and•intact•nuclei/•nuclear•pro-
    E
    teins•from•mammalian•cells•or•tissue


Benefits
 •• No•cross-contamination•of•cytoplasmic•and•nuclear•fractions
 •• • he•kit•provides•reagents•sufficient• for•extraction•of•cytoplasmic•
    T                                                                           Kit contents
    and•nuclear•fractions•from•50•samples•of•50•µl•wet•cell•pellet•or•
                                                                                Contents                                                      Size
    100•mg•of•tissue
                                                                                Cell•Lysis•Buffer                                             25•ml
                                                                                Nuclei•Washing•Buffer                                         3•x•25•ml
Technology
                                                                                Nuclei•Storage•Buffer                                         7.5•ml
Proteins• extracted• using• this•reagent• are•non-denatured,• functionally•     Nuclei•Lysis•Reagent                                          0.8•ml
active•and•therefore•ideal•for•direct•use•in•many•common•downstream•
applications,•including•1D•and•2D•electrophoresis,•Western•blotting,•
electrophoretic•mobility•shift•assay•(EMSA),•immunoprecipitation,•affinity•
                                                                                Ordering information
purification,•enzymatic•activity•and•reporter•gene•assays.•Isolated•proteins•   Order•number        Product                                          Size
are•compatible•with•the•TOTAL•protein•assay•reagent•(see• •page 87).
                                                                                P07114              DUALXtract•nuclear•and•cytoplasmic•protein•      1•kit
                                                                                                    extraction•kit




                                                                                                                                                             85
6.2.1     Protein•extraction•|•Extraction•from•fungal•and•bacterial•cells•|•TOTAL protein extraction kit




             TOTAL protein extraction kit
             ›› Extraction of total protein from fungal and bacterial cells


        Applications                                                                   Kit contents
        •• Extraction•of•total•protein•from•fungal•and•bacterial•cells                  Contents                                          Size
        •• • uitable•for•protein•extraction•of•DUALhybrid•(yeast•two-hybrid)•
           S                                                                            Extraction•Buffer•H                               20•ml
           DUALmembrane•and•DUALhunter•baits
                                                                                        Sample•Buffer                                     3•ml
                                                                                        Glass•beads                                       12•ml

        Benefits
                                                                                       Ordering information
        •• Complex•detergent•mix•guarantees•improved•protein•solubilization
        •• • nhanced• extraction• of• strongly• hydrophobic• proteins• such• as•
           E                                                                            Order•number       Product                        Size
           membrane•proteins                                                            P01013             TOTAL•protein•extraction•kit   1•kit
        •• Sufficient•for•60•protein•extractions


        Technology
        The•TOTAL•protein•extraction•kit•uses•mechanical•disruption•of•the•cell•
        wall•by•glass•beads•followed•by•protein•solubilization•with•a•complex•
        detergent•mix•to•achieve•quantitative•recovery•of•proteins•with•widely•
        different•characteristics,•including•strongly•hydrophobic•proteins•and•
        integral•membrane•proteins.

        For•total•protein•extraction•from•yeasts,•the•Lytic•Agent•may•be•used•for•
        efficient•removal•of•the•cell•wall,•please• see page 52.


  86
                                                             Protein•extraction•|•Protein•concentration•measurement•|•TOTAL protein assay reagent   6.3.1




 TOTAL protein assay reagent
 ›› Quick and easy measurement of protein concentrations from cell extracts and purified protein samples


Applications                                                                 Ordering information
•• • oncentration•measurement•of•protein•samples•using•standard•96•
   C                                                                         Order•number   Product                                      Size
   well•plates•or•1•ml•cuvettes                                              P07120         TOTAL•protein•assay•reagent                  100•ml



Benefits
•• Save•up•to•30%•cost•compared•to•standard•Bradford•assays
•• • he•reagent•is•sufficient•for•2’500•assays•(0.2•ml/•assay•using•micro•
   T
   plate)•or•500•assays•(1•ml/•assay•using•1•ml•cuvette).


Technology
The•TOTAL•protein•assay•reagent•is•based•on•the•Bradford•principle.•
Proteins•bind•to•Coomassie•Brilliant•Blue•in•an•acidic•solution•and•ab-
sorb•the•light•at•a•wavelength•of•595•nm.•The•assay•is•quick•and•easy:•
simply•mix•the•diluted•reagent•with•1/10•volume•of•the•protein•solution•
and•read•the•light•absorbance•at•595•nm.•Standard•clear•96-well•plates•
or•cuvettes•can•be•used•for•absorbance•reading.




                                                                                                                                                    87
7.1.1    Protein•refolding•|•DUALrefold•kits•|•DUALrefold soluble protein refolding kits




           DUALrefold soluble protein refolding kits
           ›› Refolding of cytosolic and nuclear proteins


        Applications                                                                   Ordering information

        •• • uickly•identify•the•optimal•refolding•conditions•for•your•protein•
           Q                                                                           Order•number   Product                                     Size
           of•interest                                                                 P07201         DUALrefold•soluble•protein•refolding•kit•   10•spin•column•sets
        •• Suitable•for•cytosolic•and•nuclear•proteins                                 P07301         DUALrefold•refolding•column•(small)•#1      10•spin•columns
                                                                                       P07302         DUALrefold•refolding•column•(small)•#2      10•spin•columns
                                                                                       P07303         DUALrefold•refolding•column•(small)•#3      10•spin•columns
        Benefits
                                                                                       P07304         DUALrefold•refolding•column•(small)•#4      10•spin•columns
        •• • olumn-based•refolding•prevents•aggregation•and•enhances•pro-
           C                                                                           P07305         DUALrefold•refolding•column•(small)•#5      10•spin•columns
           tein•recovery                                                               P07306         DUALrefold•refolding•column•(small)•#6      10•spin•columns
        •• • hoice•of•multiple•buffers•to•optimize•refolding•conditions•for•any•
           C                                                                           P07307         DUALrefold•refolding•column•(small)•#7      10•spin•columns
           protein                                                                     P07308         DUALrefold•refolding•column•(small)•#8      10•spin•columns
                                                                                       P07309         DUALrefold•refolding•column•(small)•#9      10•spin•columns
        Refolding of cytosolic and nuclear proteins                                    P07310         DUALrefold•refolding•column•(small)•#10     10•spin•columns
                                                                                       P07401         DUALrefold•refolding•column•(large)•#1      4•columns
        Test•up•to•10•different•refolding•conditions•using•the•DUALrefold•soluble•
                                                                                       P07402         DUALrefold•refolding•column•(large)•#2      4•columns
        protein•refolding•kit•to•determine•the•optimal•refolding•buffer•and•column.•
                                                                                       P07403         DUALrefold•refolding•column•(large)•#3      4•columns
        Then•use•this•buffer/•column•combination•for•preparative•large-scale•
        refolding•of•your•protein.                                                     P07404         DUALrefold•refolding•column•(large)•#4      4•columns
                                                                                       P07405         DUALrefold•refolding•column•(large)•#5      4•columns
        Small•scale•columns•(P07301•—•P07310)•are•designed•for•preparative•            P07406         DUALrefold•refolding•column•(large)•#6      4•columns
        refolding•of•0.6•—•1.2•mg•protein.                                             P07407         DUALrefold•refolding•column•(large)•#7      4•columns
                                                                                       P07408         DUALrefold•refolding•column•(large)•#8      4•columns
        Large•scale•columns•(P07401•—•P07410)•are•designed•for•preparative•            P07409         DUALrefold•refolding•column•(large)•#9      4•columns
        refolding•of•5-10•mg•protein.                                                  P07410         DUALrefold•refolding•column•(large)•#10     4•columns

  88
                                                                        Protein•refolding•|•DUALrefold•kits•|•DUALrefold membrane protein refolding kits   7.1.2




DUALrefold membrane protein refolding kits
›› Refolding of integral membrane and membrane-associated proteins


Applications                                                                       Order•   Product                                    Size
•• • uickly•identify•the•optimal•refolding•conditions•for•your•membrane•
   Q                                                                               number
   protein•of•interest                                                             P07601   DUALrefold•membrane•refolding•column•#1    4•columns
•• Suitable•for•membrane•and•membrane-associated•proteins                          P07602   DUALrefold•membrane•refolding•column•#2    4•columns
                                                                                   P07603   DUALrefold•membrane•refolding•column•#3    4•columns
Benefits                                                                           P07604   DUALrefold•membrane•refolding•column•#4    4•columns
                                                                                   P07605   DUALrefold•membrane•refolding•column•#5    4•columns
•• • olumn-based•refolding•prevents•aggregation•and•enhances•protein•
   C
                                                                                   P07606   DUALrefold•membrane•refolding•column•#6    4•columns
   recovery
•• • hoice•of•multiple•buffers•to•optimize•refolding•conditions•for•any•protein•
   C                                                                               P07607   DUALrefold•membrane•refolding•column•#7    4•columns
                                                                                   P07608   DUALrefold•membrane•refolding•column•#8    4•columns
Refolding of membrane and membrane-associated proteins                             P07609   DUALrefold•membrane•refolding•column•#9    4•columns
Test•up•to•20•different•refolding•conditions•using•the•DUALrefold•mem-             P07610   DUALrefold•membrane•refolding•column•#10   4•columns
brane• protein• refolding• kit• to• determine• the• optimal• refolding• buffer•    P07611   DUALrefold•membrane•refolding•column•#11   4•columns
and•column.•Then•use•this•buffer/•column•combination•for•preparative•              P07612   DUALrefold•membrane•refolding•column•#12   4•columns
large-scale•refolding•of•your•protein.                                             P07613   DUALrefold•membrane•refolding•column•#13   4•columns
Large•scale•columns•(P07601•—•P07610)•are•designed•for•preparative•
                                                                                   P07614   DUALrefold•membrane•refolding•column•#14   4•columns
refolding•of•5•—•10•mg•protein.
                                                                                   P07615   DUALrefold•membrane•refolding•column•#15   4•columns
                                                                                   P07616   DUALrefold•membrane•refolding•column•#16   4•columns
Ordering information
                                                                                   P07617   DUALrefold•membrane•refolding•column•#17   4•columns
 Order•     Product                                        Size
                                                                                   P07618   DUALrefold•membrane•refolding•column•#18   4•columns
 number
                                                                                   P07619   DUALrefold•membrane•refolding•column•#19   4•columns
 P07501     DUALrefold•membrane•protein•refolding•kit      20•spin•column•sets
                                                                                   P07620   DUALrefold•membrane•refolding•column•#20   4•columns
                                                                                                                                                           89
7.2.1    Protein•refolding•|•Protein•stability•and•aggregation•assays•|•Protein stability and aggregation assay kit




           Protein stability and aggregation assay kit
           ›› Evaluation of protein thermal stability and analysis of protein unfolding


        Applications                                                                        Kit contents
        •• • nzymatic•assay•to•determine•the•thermal•stability•and•unfolding•
           E                                                                                Contents                                                        Size
           of•your•protein•of•interest                                                      1000•x•PSA•dye                                                  60•µl


                                                                                            Ordering information
        Benefits
        •• Determine•the•optimal•refolding•conditions•for•any•denaturated•protein•
                                                                                 •          Order•number     Product                                        Size
        •• • ach•kit•contains•reagent•for•200•assays•with•an•assay•volume•of•
           E                                                                                P07621           Protein•stability•and•aggregation•assay•kit•   1•kit
           0.3•ml•using•96-well•plates•or•cuvettes.


        Technology
        The•Protein•stability•and•aggregation•assay•kit•determines•the•thermal•stability•
        of•a•protein,•as•well•as•its•folding•and•unfolding•characteristics.•The•assay•
        is•based•on•a•fluorescent•dye•which•binds•to•the•hydrophobic•surfaces•of•
        proteins•and•generates•a•fluorescent•signal•at•635•nm.•Since•unfolded•proteins•
        have•more•exposed•hydrophobic•surfaces•than•native•proteins,•unfolded•
        proteins•generate•a•stronger•fluorescent•signal•than•folded•proteins.•The•kit•
        allows•determination•of•thermal•unfolding•curves•and•calculation•of•protein•
        half•life•times•(t1/2),•which•in•turn•can•be•used•to•evaluate•protein•stability.•
        Thermal•unfolding•curves•can•also•be•used•to•analyze•protein•unfolding•and•
        aggregation•states.


  90
                                                Kits•and•reagents•for•sample•preparation•|•QuickSpin protein concentration/ buffer exchange/ desalting kit                   8.1.1




 QuickSpin protein concentration/ buffer exchange/ desalting kit
 ›› Efficient concentration of dilute protein solutions, for buffer exchange, and for removing different types of salts from protein samples


Applications
•• • imultaneous•removal•of•salts•and•concentration•of•dilute•protein•
   S
   solutions
•• Preparation•of•proteins•for•downstream•applications

                                                                                             Kit contents
Benefits
•• • ach•spin•column•is•able•to•concentrate•and•desalt•up•to•50•μg•of•
   E                                                                                         Contents                                                         Size
   acidic•or•basic•proteins                                                                  Column•equilibration•and•wash•buffer•(acidic)                    30•ml
•• Quick•and•easy•to•use,•no•need•for•dialysis•or•precipitation•steps                        Column•activation•and•wash•buffer•(basic)                        30•ml
                                                                                             pH•binding•buffer•(acidic)                                       3•ml
Technology                                                                                   pH•binding•buffer•(basic)                                        3•ml
                                                                                             Elution•buffer                                                   4•ml
                                                             The•kit•utilizes•spin•col-
                                                             umns,• which• bind• the•        Neutralizer                                                      1•ml
                                        Follow the procedure                                 Micro•spin•columns                                               25•pcs
                                        for basic proteins   protein•of•interest•if•it•re-
                                                             tains•a•net•positive•char-      Collection•tubes                                                 25•pcs
            2       4       6               10      12       ge.•Nonspecifically•bound•      Elution•tubes•(1.7•ml)                                           25•pcs
         Follow the procedure
         for acidic proteins
                                                             materials• such• as• salts•
                                                             are•washed•from•the•col-
                                                                                             Ordering information
                                                             umn• and• the• specific•
                             Isoelectric Point
                                                             protein• is• eluted• into• a•   Order•number       Product                                              Size
small•volume•of•elution•buffer.•The•process•results•in•an•effective•con-                     P07701             QuickSpin•protein•concentration/•buffer•ex-          1•kit
centration•and•desalting•of•the•protein.                                                                        change/•desalting•kit•
                                                                                                                                                                             91
8.1.2     Kits•and•reagents•for•sample•preparation•|•QuickSpin detergent removal kit




            QuickSpin detergent removal kit
            ›› Quick and efficient removal of detergents in protein solutions


        Applications
         •• • emoval•of•all•types•of•detergents•including•ionic,•non-ionic•and•
            R
            zwitterionic•detergents.•
         •• • esigned•to•remove•detergents•in•protein•solutions•either•in•their•
            D
            free•form•or•complexed•with•proteins
                                                                                                           Kit contents
        Benefits
                                                                                                               Contents                                              Size
         •• More•than•95%•of•detergents•from•protein•samples•removed                                           Column•activation•and•wash•buffer•(acidic)            8•ml
         •• Easy•to•handle•Micro•Spin•Columns•guarantee•a•high•protein•recovery
                                                                                                               Column•activation•and•wash•buffer•(basic)             8•ml
         •• • ach• column• removes• detergents• from• up• to• 50• µg• of• acidic• or•
            E
                                                                                                               pH•binding•buffer•(acidic)                            4•ml
            basic•total•protein
                                                                                                               pH•binding•buffer•(basic)                             4•ml
        Technology                                                                                             Elution•buffer                                        4•ml
                                                                      The•kit•utilizes•spin•columns,•          Neutralizer                                           1•ml
                                                                      which• bind• the• protein• of•           Micro•spin•columns                                    25•pcs
                                                 Follow the procedure
                                                 for basic proteins   interest• if• it• retains• a• net•       Collection•tubes                                      25•pcs
                                                                      positive•charge.•Non-specif-
                                                                                                               Elution•tubes•(1.7•ml)                                25•pcs
                     2       4       6               10      12       ically• bound• proteins• and•
                  Follow the procedure
                                                                      detergents•are•washed•from•          •              •
                  for acidic proteins
                                                                      the•column•and•the•specific•         Ordering information
                                                                      protein•is•eluted•into•a•spe-
                                      Isoelectric Point
                                                                      cially• formulated• elution•             Order•number       Product                             Size
        buffer,•removing•the•detergent•from•the•protein•sample.•Each•column•                                   P07702             QuickSpin•detergent•removal•kit•    1•kit
        will•remove•detergent•from•up•to•50•µg•of•acidic•or•basic•total•protein.
  92
                                                                                                                                            Literature    9




Literature
›› Complete list of publications reporting the successful use of Dualsystems’ protein interaction systems

More•than•60•publications• in•peer•reviewed• journals• with•an•average•
                                                                               Mutations in the LRRK2 Roc-COR tandem domain link Parkinson‘s
impact•factor•of•9.6•show•the•high•impact•of•our•technologies!                 disease to Wnt signalling pathways.•Sancho•et•al.•Hum. Mol. Genet.,•
                                                                               18:•3955•-•3968.•(2009)
 SH3TC2, a protein mutant in Charcot–Marie–Tooth neuropathy,                   FE65 Binds Teashirt, Inhibiting Expression of the Primate- Specific
 links peripheral nerve myelination to endosomal recycling.•Stendel•           Caspase-4.•Kajiwara•et•al.•PLOS one•4(4):e5071,•1-17•(2009)
 et•al.•Brain•133:•2462•–•2474•(2010)
                                                                               Memo is a cofilin-interacting protein that influences PLC 1 and
 A remorin protein interacts with symbiotic receptors and regulates            cofilin activities, and is essential for maintaining directionality
 bacterial infection. Lefebvre•et•al.•PNAS,•107:•2343•-•2348.•(2010)           during ErbB2-induced tumor-cell migration.• Meira• et• al.• J. Cell
 Tmem176B and Tmem176A are associated with the immature                        Sci.,•122:•787•-•797.•(2009)
 state of dendritic cells.•Condamine•et•al.•J.Leukoc. Biol.•10.1189/           Nyctalopin Interacts with Transient Receptor Potential Channels
 jlb.1109738.•(2010)                                                           in Yeast. Bojang•et•al.•Invest. Ophthalmol. Vis. Sci.,•50:•5176.(2009)
 Cockayne syndrome group B protein promotes mitochondrial                      Pex3 peroxisome biogenesis proteins function in peroxisome
 DNA stability by supporting the DNA repair association with the               inheritance as class V myosin receptors.•Chang•et•al.•J Cell Biol.
 mitochondrial membrane. Aamann•et•al.•FASEB J,•10.1096/fj.09-                 187(2):233-46•(2009)
 147991.•(2010)
                                                                               MS4A4B Is a GITR-Associated Membrane Adapter, Expressed
 D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains                 by Regulatory T Cells, Which Modulates T Cell Activation. Howie•
 and Regulates Transferrin Receptor Recycling.•Eggers•et•al.•J. Biol.          et•al.•Journal of Immunology•183:•4197–4204.•(2009)
 Chem.,•284:•32869•-•32880.•(2009)
                                                                               Hypoxia Inducible Factor-2 Stabilization and Maxi-K+ Channel
 Biogenesis of cytochrome b6 in photosynthetic membranes.•                     α1-Subunit Gene Repression by Hypoxia in Cardiac Myocytes: Role
 Saint-Marcoux•et•al.•J Cell Biol.•185(7):1195-207•(2009)                      in Preconditioning. Bautista•et•al.•Circ. Res.•104:•1364•-•1372.•(2009)
 KDP-1 is a nuclear envelope KASH protein required for cell-cycle              Heme Oxygenase-2 and Large-Conductance Ca2+-activated K+
 progression.•McGee•et•al.•J. Cell Sci.,•122:•2895•-•2905.•(2009)              Channels: Lung Vascular Effects of Hypoxia.•Roth•et•al.•Am. J.
 A Bacterial-Type ABC Transporter Is Involved in Aluminum Toler-               Respir. Crit.•Care•Med.,•180:•353•-•364.•(2009)
 ance in Rice. Huang•et•al.•PLANT CELL,•21:•655•-•667.•(2009)

                                                                                                                                                         93
9    Literature




     Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6             SLITRK1 binds 14-3-3 and regulates neurite outgrowth in a
     Interact with Cytochrome b5 to Regulate Their Affinity for Substrate                                                                       •
                                                                              phosphorylation-dependent manner. Kajiwara•et•al.•Biol Psychiatry.•
     Sugars. Fan•et•al.•Plant Physiol. (2009)                                 66(10):918-25•(2009)
     EHD4 AND CDH23 ARE INTERACTING PARTNERS IN COCHLEAR                      Cdc34p ubiquitin-conjugating enzyme is a component of the
     HAIR CELLS. Sengupta•et•al.•JBC•284:•20121•-•20129.•(2009)               tombusvirus replicase complex and ubiquitinates p33 replication
     Identification of small subunits of mammalian serine palmitoyl-          protein.•Li•et•al.•J.Virol.•82(14):6911-26•(2008)
     transferase that confer distinct acyl-CoA substrate specificities.•      Lotus japonicus CASTOR and POLLUX Are Ion Channels Essential
     Han•et•al.•PNAS,•106:•8186•-•8191.•(2009)                                for Perinuclear Calcium Spiking in Legume Root Endosymbiosis.•
     Physical and Functional Interaction between the Dopamine Trans-          Charpentier•et•al.•PLANT CELL,•20:•3467•-•3479.•(2008)
     porter and the Synaptic Vesicle Protein Synaptogyrin-3.•Egaña•et•        The Cyanobacterial Homologue of HCF136/YCF48 Is a Component of
     al.•J. Neurosci.,•29:•4592•-•4604.•(2009)                                an Early Photosystem II Assembly Complex and Is Important for Both
     The Cyanobacterial Homologue of HCF 136/YCF48 Is a Component             the Efficient Assembly and Repair of Photosystem II in Synechocystis
     of an Early Photosystem II Assembly Complex and Is Important             sp. PCC 6803. Komenda•et•al.•J. Biol. Chem.;•283:•22390•-•22399.•(2008)
     for Both the Efficient Assembly and Repair of Photosystem II in          Monitoring Protein-Protein Interactions between the Mammalian
     Synechocystis sp. PCC•6803.•Komenda•et•al.•JBC•283:33•(2009)             Integral Membrane Transporters and PDZ-interacting Partners
     Identification of ERGIC-53 as an intracellular transport receptor        Using a Modified Split-ubiquitin Membrane Yeast Two-hybrid
     of alpha1-antitrypsin.Nyfeler•et•al.•J Cell Biol.•180(4):705-12•(2009)   System.•Gisler•et•al.•Mol. Cell. Proteomics,•7:•1362•–•1377•(2008)

     Vascular Effects of Heme Oxygenase-2 and the BK-Channel in               Interaction assays in yeast and cultured cells confirm known and
     Acute, Sustained, and Chronic Alveolar Hypoxia.•Roth•et•al.•Am.          identify novel partners of the synaptic vesicle protein synapto-
     J. Respir. Crit. Care Med.,•179:•A6252•(2009)                            physin.•Felkl•et•al.•Neuroscience•156:•344-352•(2008)

     Identifying components of the hair-cell interactome involved in          Negative regulation of the yeast ABC transporter YCF1P by
     cochlear amplification. Zheng•et•al.•BMC Genomics•10:•127•(2009)         phosphorylation within its N-terminal extension.•Paumi•et•al.•J.
                                                                              Biol. Chem. (2008)•Jul•29
     Interactions between membrane-bound cellulose synthases
     involved in the synthesis of the secondary cell wall.•Timmers•et•        Identification of ERGIC-53 as an intracellular transport receptor
     al.•FEBS Letters•583:•978-982•(2009)                                     of 1-antitrypsin.•Nyfeler•et•al.•J. Cell Biol.,•180:•705•-•712.•(2008)

     Analysis of the Chloroplast Protein Kinase Stt7 during State Tran-       LPA2 Is Required for Efficient Assembly of Photosystem II in Ara-
     sitions. Lemeille•et•al.•PloS Biol.•7(3):•e1000045•(2009)                bidopsis thaliana. Ma•et•al.•PLANT CELL,;•19:•1980•-•1993.•(2007)


94
                                                                                                                                       Literature      9




Mapping protein-protein interactions for the yeast ABC transporter    Low PSII Accumulation Is Involved in Efficient Assembly of
Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid         Photosystem II in Arabidopsis thaliana. Peng,•L.•et•al.•Plant Cell•
analysis.•Paumi•et•al.•Mol. Cell 26(1):15-25•(2007)•                  18(4):955-969•(2006)•
Brain-type creatine kinase BB-CK interacts with the Golgi Matrix      G Protein-coupled Receptor Gpr4 Senses Amino Acids and Ac-
Protein GM130 in early prophase.•Burklen•et•al.•Mol. Cell. Biochem.   tivates the cAMP-PKA Pathway in Cryptococcus neoformans.
297,•53-64•(2007)                                                     Xue,•C.•et•al.•Mol. Biol. Cell 17(2):667-679•(2006)•
CCM3 interacts with CCM2 indicating common pathogenesis               The familial dementia BRI2 gene binds the Alzheimer‘s gene APP
for cerebral cavernous malformations.•Voss•et•al.•Neurogenetics•      and inhibits Abeta production. •Matsuda,•S.•et•al.•J. Biol. Chem.
8:249–256•(2007)                                                      280:•28912-28916•(2005)•
Molecular dissection of NRG1-ERBB4 signaling implicates PTPRZ1        Large-scale identification of yeast integral membrane protein interac-
as a potential schizophrenia susceptibility gene. Buxbaum•et•al.•     tions. Miller,•J.P.•et•al.•Proc. Natl. Acad. Sci USA•102:•12123-12128•(2005)•
Mol. Psychiatry. Apr•17•(2007)•                                       Promyelocytic leukemia zinc finger protein localizes to the coch-
XLF Interacts with the XRCC4-DNA Ligase IV Complex to Promote         lear outer hair cells and interacts with prestin, the outer hair cell
DNA Nonhomologous End-Joining.•Ahnesorg•et•al.•Cell•124(2),•301-      motor protein.•Nagy•et•al.•Hearing Research•204,•216-222•(2005)•
313•(2006)                                                            Erg28p is a key protein in the yeast sterol biosynthetic enzyme
Medaka simplet (FAM53B) belongs to a family of novel vertebrate       complex. Mo•C.,•Bard,•M.•J.•Lipid Res.•46:•1991-1998•(2005)•
genes controlling cell proliferation. Thermes•et•al.•Development,•    The yeast split-ubiquitin system to study chloroplast membrane
133:•1881•-•1890.•(2006)                                              protein interactions.•Pasch,•J.C.,•Nickelsen,•J.,•Schünemann,•D.•
Calsyntenin-1 Docks Vesicular Cargo to Kinesin-1. Konecna•et•al.•     Appl.•Microbiol. Biotechnol.•69:•440-447•(2005)•
Molecular Biology of the Cell 17,Issue•8,•3651-3663•(2006)            Studies of yeast oligosaccharyl transferase subunits using the split-
UNC-83 IS a KASH protein required for nuclear migration and is        ubiquitin system: topological features and in vivo interactions. Yan,•A.,•
recruited to the outer nuclear membrane by a physical interac-        Wu,•E.,•Lennarz,•W.J.•Proc.•Natl. Acad. Sci USA 102(20):7121-7126•(2005)•
tion with the SUN protein UNC-84. McGee• et• al.• Mol. Bio.l Cell     Subunits of the translocon interact with components of the oli-
17(4):1790-801•(2006)•                                                gosaccharyl transferase complex.•Chavan,•M.M.,•Yan,•A.,•Lennarz,•
                                                                  •
Analysis of the oligomeric structure of the motor protein prestin.•   W.J.••J.•Biol. Chem.•280:•22917-22924•(2005)•
Zheng,•J.•et•al.•J. Biol. Chem.•281(29):19916-19924•(2006)•           NEDD8 Ultimate Buster-1L Interacts with the Ubiquitin-like Protein
                                                                      FAT10 and Accelerates Its Degradation. Hipp•et•al.•J. Biol. Chem.•279•
                                                                      (16),•16503-16510•(2004)•
                                                                                                                                                      95
9    Literature




     The ERG28-encoded protein, Erg28p, interacts with both the sterol
                                                                               Reviews about the DUALmembrane system
     C-4 demethylation enzyme complex as well as the late biosynthetic
     protein, the C-24 sterol methyltransferase (Erg6p).•Mo,•C.,•Valacho-
     vic,•M.,•and•Bard,•M.•Biochim. Biophys. Acta•1686(1-2):30-6•(2004)•       Membrane-based yeast two-hybrid system to detect protein
                                                                               interactions. Lentze•et•al.•Curr Protoc Protein Sci, Chapter 19:•Unit•
     Use of the Split-Ubiquitin Two-Hybrid System to Identify Proteins
                                                                               19.17.•(2008)
     Interacting With the Alzheimer Proteins APP and LRP. •Vitale,•R.•
     and•Buxbaum,•JD.•Biol. Bull.•207(2):167•(2004)•                           Methods for mapping of interaction networks involving membrane
                                                                               proteins. Hooker•B.S.,•Bigelow•D.J.,•Lin•C.T.•Biochem. Biophys. Res.
     The yeast split-ubiquitin membrane protein two-hybrid screen
                                                                               Commun.•Sep•19•(2007)•epub•ahead•of•print.•
     identifies BAP31 as a regulator of the turnover of endoplasmic
     reticulum-associated protein tyrosine phosphatase-like B. Wang•           Yeast-based functional genomics and proteomics technologies:
     et•al.•Mol. Cell Biol. 24(7):2767-78•(2004)•                              the first 15 years and beyond.•Suter,•B.,•Auerbach,•D.•and•Stagljar,•
                                                                               I.•Biotechniques•40(5),•625-644•(2006).•
     The Arabidopsis putative G protein-coupled receptor GCR1 interacts
     with the G protein alpha subunit GPA1 and regulates abscisic acid         Utilizing the Split-Ubiquitin Membrane Yeast Two-Hybrid System
     signaling. Pandey,•S.•and•Assmann,•SM.•Plant Cell 16(6):1616-32•(2004)•   to Identify Protein-Protein Interactions of Integral Membrane
                                                                               Proteins.•Iyer•et•al.•Sci. STKE,•2005:•pl3.
     Identification of Novel ErbB3-interacting Factors Using the Split-
     Ubiquitin Membrane Yeast Two-Hybrid System.• Thaminy• et• al.•            Proteomic approaches for generating comprehensive protein
     Genome Research•13:1744-53•(2003)•                                        interaction maps. Auerbach,•D.,•Fetchko,•M,•and•Stagljar,•I.•Targets
                                                                               (Drug Discovery Today)•2,•85-92•(2003)•
     A genetic system based on split-ubiquitin for the analysis of
     interactions between membrane proteins in vivo. Stagljar•et•al.•          The post-genomic era of interactive proteomics: Facts and per-
     Proc. Natl. Acad. Sci.•95,•5187-5192•(1998)                               spectives. Auerbach,•D.,•Thaminy,•S.,•Hottiger,•M.,•and•Stagljar,•I.•
                                                                               Proteomics•2,•611-623•(2002)•
     Original publications using the DUALhunter system                         Genetic approaches to the identification of interactions between
     .                                                                         membrane proteins in yeast. Auerbach,•D.,•Galeuchet-Schenk,•B.,•
     Yeast split-ubiquitin-based cytosolic screening system to detect          Hottiger,•M.O.,•and•Stagljar,•I.•J.•Recept.•Signal Transduct. Res.•22,•
     interactions between transcriptionally active proteins.•Möckli•et•        473-483•(2002)•
     al.•BioTechniques•42(6):725-730•(2007)                                    Analysis of membrane protein interactions using yeast-based
                                                                               technologies.• Stagljar,• I.• and• Fields,• S.• Trends Biochem.• Sci.• 27,•
                                                                               559-563•(2002)•

96
                                                                                                                                License information           10




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 98
                                                                                                                              Alphabetical product listing        12




                                                                             DUALhunter•starter•kit•+•Human•adult•brain•cDNA•library•(NubG-x)               44
Alphabetical product listing                                                 DUALhunter•starter•kit•+•Human•adult•brain•cDNA•library•(x-NubG)               44
                                                                             DUALhunter•starter•kit•+•Human•adult•colon•cDNA•library•(NubG-x)               44
-A-                                                                          DUALhunter•starter•kit•+•Human•adult•kidney•cDNA•library•(NubG-x)              44
96-well•anionic•protein•binding•plate•(black)                           65   DUALhunter•starter•kit•+•Human•adult•kidney•cDNA•library•(x-NubG)              44
96-well•anionic•protein•binding•plate•(transparent)                     65   DUALhunter•starter•kit•+•Human•adult•liver•cDNA•library•(NubG-x)               44
96-well•cationic•protein•binding•plate•(black)                          65   DUALhunter•starter•kit•+•Human•adult•lung•cDNA•library•(NubG-x)                44
96-well•cationic•protein•binding•plate•(transparent)                    65   DUALhunter•starter•kit•+•Human•embryonal•brain•cDNA•library•(NubG-x)           44
A. thaliana•cDNA•library•(NubG-x)                                       36   DUALhunter•starter•kit•+•Jurkat•T•cells••cDNA•library•(NubG-x)                 44
A. thaliana•cDNA•library•(pGAD-DS)                                      15   DUALhunter•starter•kit•+•LNCaP•cells•cDNA•library•(NubG-x)                     44
                                                                             DUALhunter•starter•kit•+•Mammary•epithelial•cells•cDNA•library•(NubG-x)        44
-C-
                                                                             DUALhunter•starter•kit•+•Medicago•nodules•cDNA•library•(NubG-x)                44
C. elegans•whole•adult•cDNA•library•(NubG-x)                            36
                                                                             DUALhunter•starter•kit•+•Medicago•nodules•cDNA•library•(x-NubG)                44
C. elegans•whole•eggs•DNA•library•(NubG-x)                              36
                                                                             DUALhunter•starter•kit•+•Mouse•adult•heart•cDNA•library•(NubG-x)               44
C. elegans•whole•adult•cDNA•library•(pGAD-DS)                           15
                                                                             DUALhunter•starter•kit•+•Mouse•adult•kidney•cDNA•library•(NubG-x)              44
Custom•normalized•cDNA•library•construction                             62
                                                                             DUALhunter•starter•kit•+•Mouse•adult•spleen•cDNA•library•(x-NubG)              44
Custom•standard•cDNA•library•construction                               62
                                                                             DUALhunter•starter•kit•+•Mouse•whole•embryo,•11•days•cDNA•library•(NubG-x)     44
-D-                                                                          DUALhunter•starter•kit•+•Normalized•human•fetal•brain•cDNA•library••(NubG-x)   44
D. melanogaster•embryo•cDNA•library•(NubG-x)                            36   DUALhunter•starter•kit•+•Normalized•human•spleen•cDNA•library•(NubG-x)         44
D. melanogaster•cDNA•library•(NubG-x)                                   36   DUALhunter•starter•kit•+•Normalized•mouse•brain•cDNA•library•(NubG-x)          44
DS•Yeast•transformation•kit                                             48   DUALhunter•starter•kit•+•Normalized•mouse•embryo•cDNA•library•(x-NubG)         44
DSY-5•yeast•expression•strain                                           78   DUALhunter•starter•kit•+•Rat•neonatal•cardiomyocyte•cDNA•library•(NubG-x)      44
DSY-6•yeast•expression•strain                                           78   DUALhunter•starter•kit•+•S. cerevisiae•cDNA•library•(NubG-x)                   44
DUALhunter•control•plasmid•set•(3•controls)                             45   DUALhunter•starter•kit+•Mouse•adult•heart•cDNA•library•(x-NubG)                44
DUALhunter•screening•services                                           39   DUALhybrid•control•plasmid•set•(3•controls)                                    14
DUALhunter•starter•kit•+•Mouse•adult•brain•cDNA•library•(NubG-x)        44   DUALhybrid•kit                                                                 12
DUALhunter•starter•kit•+•A. thaliana•cDNA•library•(NubG-x)              44   DUALhybrid•service,•bait•dependency•test•(10•interactions)                     11
DUALhunter•starter•kit•+•C. elegans•whole•adult•cDNA•library•(NubG-x)   44   DUALhybrid•service,•pairwise•interaction•(10•interactions)•                    11
DUALhunter•starter•kit•+•C. elegans•whole•eggs•DNA•library•(NubG-x)     44   DUALhybrid•services•Gold                                                        8
DUALhunter•starter•kit•+•D. melanogaster•embryo•cDNA•library•(NubG-x)   44   DUALhybrid•services•Platinum                                                    8
DUALhunter•starter•kit•+•D. melanogaster•cDNA•library•(NubG-x)          44   DUALhybrid•services•Silver                                                      8
DUALhunter•starter•kit•+•HeLa•cell•line•cDNA•library•(NubG-x)           44   DUALmembrane•control•plasmid•set•(3•controls)                                  32

                                                                                                                                                                 99
12      Alphabetical product listing


      DUALmembrane•false•positive•filtering•service                                      25   DUALmembrane•starter•kit•N•+•S. cerevisiae•cDNA•library•(NubG-x)                     25
      DUALmembrane•pairwise•interaction•kit                                              30   DUALmembrane•starter•kit•STE•+•A. thaliana•cDNA•library•(NubG-x)                     29
      DUALmembrane•screening•services                                                    18   DUALmembrane•starter•kit•STE•+•C. elegans•whole•adult•cDNA•library•(NubG-x)          29
      DUALmembrane•starter•kit•N•+•A. thaliana•cDNA•library•(NubG-x)                     25   DUALmembrane•starter•kit•STE•+•C. elegans•whole•eggs•DNA•library•(NubG-x)            29
      DUALmembrane•starter•kit•N•+•C. elegans•whole•adult•cDNA•library•(NubG-x)          25   DUALmembrane•starter•kit•STE•+•D. melanogaster embryo•cDNA•library•(NubG-x)          29
      DUALmembrane•starter•kit•N•+•C. elegans•whole•eggs•DNA•library•(NubG-x)            25   DUALmembrane•starter•kit•STE•+•D. melanogaster•cDNA•library•(NubG-x)                 29
      DUALmembrane•starter•kit•N•+•D. melanogaster•embryo•cDNA•library•(NubG-x)          25   DUALmembrane•starter•kit•STE•+•HeLa•cell•line•cDNA•library•(NubG-x)                  28
      DUALmembrane•starter•kit•N•+•D. melanogaster•cDNA•library•(NubG-x)                 25   DUALmembrane•starter•kit•STE•+•Human•adult•brain•cDNA•library•(NubG-x)               28
      DUALmembrane•starter•kit•N•+•HeLa•cell•line•cDNA•library•(NubG-x)                  24   DUALmembrane•starter•kit•STE•+•Human•adult•brain•cDNA•library•(x-NubG)               28
      DUALmembrane•starter•kit•N•+•Human•adult•brain•cDNA•library•(NubG-x)               24   DUALmembrane•starter•kit•STE•+•Human•adult•colon•cDNA•library•(NubG-x)               28
      DUALmembrane•starter•kit•N•+•Human•adult•brain•cDNA•library•(x-NubG)               24   DUALmembrane•starter•kit•STE•+•Human•adult•kidney•cDNA•library•(NubG-x)              28
      DUALmembrane•starter•kit•N•+•Human•adult•colon•cDNA•library•(NubG-x)               24   DUALmembrane•starter•kit•STE•+•Human•adult•kidney•cDNA•library•(x-NubG)              28
      DUALmembrane•starter•kit•N•+•Human•adult•kidney•cDNA•library•(NubG-x)              24   DUALmembrane•starter•kit•STE•+•Human•adult•liver•cDNA•library•(NubG-x)               28
      DUALmembrane•starter•kit•N•+•Human•adult•kidney•cDNA•library•(x-NubG)              24   DUALmembrane•starter•kit•STE•+•Human•adult•lung•cDNA•library•(NubG-x)                28
      DUALmembrane•starter•kit•N•+•Human•adult•liver•cDNA•library•(NubG-x)               24   DUALmembrane•starter•kit•STE•+•Human•embryonal•brain•cDNA•library•(NubG-x)           28
      DUALmembrane•starter•kit•N•+•Human•adult•lung•cDNA•library•(NubG-x)                24   DUALmembrane•starter•kit•STE•+•Jurkat•T•cells•cDNA•library•(NubG-x)                  28
      DUALmembrane•starter•kit•N•+•Human•embryonal•brain•cDNA•library•(NubG-x)           24   DUALmembrane•starter•kit•STE•+•LNCaP•cells••cDNA•library•(NubG-x)                    28
      DUALmembrane•starter•kit•N•+•Jurkat•T•cells•cDNA•library•(NubG-x)                  24   DUALmembrane•starter•kit•STE•+•Mammary•epithelial•cells•cDNA•library•(NubG-x)        28
      DUALmembrane•starter•kit•N•+•LNCaP•cells••cDNA•library•(NubG-x)                    24   DUALmembrane•starter•kit•STE•+•Medicago•nodules•cDNA•library•(NubG-x)                28
      DUALmembrane•starter•kit•N•+•Mammary•epithelial•cells•cDNA•library•(NubG-x)        24   DUALmembrane•starter•kit•STE•+•Medicago•nodules•cDNA•library•(x-NubG)                28
      DUALmembrane•starter•kit•N•+•Medicago•nodules•cDNA•library•(NubG-x)                24   DUALmembrane•starter•kit•STE•+•Mouse•adult•brain•cDNA•library•(NubG-x)               29
      DUALmembrane•starter•kit•N•+•Medicago•nodules•cDNA•library•(x-NubG)                24   DUALmembrane•starter•kit•STE•+•Mouse•adult•heart•cDNA•library•(NubG-x)               29
      DUALmembrane•starter•kit•N•+•Mouse•adult•brain•cDNA•library•(NubG-x)               25   DUALmembrane•starter•kit•STE•+•Mouse•adult•heart•cDNA•library•(x-NubG)               29
      DUALmembrane•starter•kit•N•+•Mouse•adult•heart•cDNA•library•(NubG-x)               25   DUALmembrane•starter•kit•STE•+•Mouse•adult•kidney•cDNA•library•(NubG-x)              29
      DUALmembrane•starter•kit•N•+•Mouse•adult•heart•cDNA•library•(x-NubG)               25   DUALmembrane•starter•kit•STE•+•Mouse•adult•spleen•cDNA•library•(x-NubG)              29
      DUALmembrane•starter•kit•N•+•Mouse•adult•kidney•cDNA•library•(NubG-x)              25   DUALmembrane•starter•kit•STE•+•Mouse•whole•embryo,•11•days•cDNA•library•(NubG-x)     29
      DUALmembrane•starter•kit•N•+•Mouse•adult•spleen•cDNA•library•(x-NubG)              25   DUALmembrane•starter•kit•STE•+•Normalized•human•fetal•brain•cDNA•library••(NubG-x)   29
      DUALmembrane•starter•kit•N•+•Mouse•whole•embryo,•11•days•cDNA•library•(NubG-x)     25   DUALmembrane•starter•kit•STE•+•Normalized•human•spleen•cDNA•library•(NubG-x)         29
      DUALmembrane•starter•kit•N•+•Normalized•human•fetal•brain•cDNA•library••(NubG-x)   25   DUALmembrane•starter•kit•STE•+•Normalized•mouse•brain•cDNA•library•(NubG-x)          29
      DUALmembrane•starter•kit•N•+•Normalized•human•spleen•cDNA•library•(NubG-x)         25   DUALmembrane•starter•kit•STE•+•Normalized•mouse•embryo•cDNA•library•(x-NubG)         29
      DUALmembrane•starter•kit•N•+•Normalized•mouse•brain•cDNA•library•(NubG-x)          25   DUALmembrane•starter•kit•STE•+•Rat•neonatal•cardiomyocyte•cDNA•library•(NubG-x)      29
      DUALmembrane•starter•kit•N•+•Normalized•mouse•embryo•cDNA•library•(x-NubG)         25   DUALmembrane•starter•kit•STE•+•S. cerevisiae•cDNA•library•(NubG-x)                   29
      DUALmembrane•starter•kit•N•+•Rat•neonatal•cardiomyocyte•cDNA•library•(NubG-x)      25   DUALmembrane•starter•kit•SUC•+•A. thaliana•cDNA•library•(NubG-x)                     27

100
                                                                                                                                             Alphabetical product listing         12


DUALmembrane•starter•kit•SUC•+•C.•elegans•whole•adult•cDNA•library•(NubG-x)          27   DUALrefold•membrane•protein•refolding•column#1                                    89
DUALmembrane•starter•kit•SUC•+•C. elegans•whole•eggs•DNA•library•(NubG-x)            27   DUALrefold•membrane•protein•refolding•column#10                                   89
DUALmembrane•starter•kit•SUC•+•D. melanogaster embryo•cDNA•library•(NubG-x)          27   DUALrefold•membrane•protein•refolding•column#11                                   89
DUALmembrane•starter•kit•SUC•+•D. melanogaster•cDNA•library•(NubG-x)                 27   DUALrefold•membrane•protein•refolding•column#12                                   89
DUALmembrane•starter•kit•SUC•+•HeLa•cell•line•cDNA•library•(NubG-x)                  26   DUALrefold•membrane•protein•refolding•column#13                                   89
DUALmembrane•starter•kit•SUC•+•Human•adult•brain•cDNA•library•(NubG-x)               26   DUALrefold•membrane•protein•refolding•column#14                                   89
DUALmembrane•starter•kit•SUC•+•Human•adult•brain•cDNA•library•(x-NubG)               26   DUALrefold•membrane•protein•refolding•column#15                                   89
DUALmembrane•starter•kit•SUC•+•Human•adult•colon•cDNA•library•(NubG-x)               26   DUALrefold•membrane•protein•refolding•column#16                                   89
DUALmembrane•starter•kit•SUC•+•Human•adult•kidney•cDNA•library•(NubG-x)              26   DUALrefold•membrane•protein•refolding•column#17                                   89
DUALmembrane•starter•kit•SUC•+•Human•adult•kidney•cDNA•library•(x-NubG)              26   DUALrefold•membrane•protein•refolding•column#18                                   89
DUALmembrane•starter•kit•SUC•+•Human•adult•liver•cDNA•library•(NubG-x)               26   DUALrefold•membrane•protein•refolding•column#19                                   89
DUALmembrane•starter•kit•SUC•+•Human•adult•lung•cDNA•library•(NubG-x)                26   DUALrefold•membrane•protein•refolding•column#2                                    89
DUALmembrane•starter•kit•SUC•+•Human•embryonal•brain•cDNA•library•(NubG-x)           26   DUALrefold•membrane•protein•refolding•column#20                                   89
DUALmembrane•starter•kit•SUC•+•Jurkat•T•cells•cDNA•library•(NubG-x)                  26   DUALrefold•membrane•protein•refolding•column#3                                    89
DUALmembrane•starter•kit•SUC•+•LNCaP•cells••cDNA•library•(NubG-x)                    26   DUALrefold•membrane•protein•refolding•column#4                                    89
DUALmembrane•starter•kit•SUC•+•Mammary•epithelial•cells•cDNA•library•(NubG-x)        26   DUALrefold•membrane•protein•refolding•column#5                                    89
DUALmembrane•starter•kit•SUC•+•Medicago•nodules•cDNA•library•(NubG-x)                26   DUALrefold•membrane•protein•refolding•column#6                                    89
DUALmembrane•starter•kit•SUC•+•Medicago•nodules•cDNA•library•(x-NubG)                26   DUALrefold•membrane•protein•refolding•column#7                                    89
DUALmembrane•starter•kit•SUC•+•Mouse•adult•whole•brain•cDNA•library•(NubG-x)         27   DUALrefold•membrane•protein•refolding•column#8                                    89
DUALmembrane•starter•kit•SUC•+•Mouse•adult•heart•cDNA•library•(NubG-x)               27   DUALrefold•membrane•protein•refolding•column#9                                    89
DUALmembrane•starter•kit•SUC•+•Mouse•adult•heart•cDNA•library•(x-NubG)               27   DUALrefold•membrane•protein•refolding•kit                                         89
DUALmembrane•starter•kit•SUC•+•Mouse•adult•kidney•cDNA•library•(NubG-x)              27   DUALrefold•soluble•protein•ref.•column•(large)#1                                  88
DUALmembrane•starter•kit•SUC•+•Mouse•adult•spleen•cDNA•library•(x-NubG)              27   DUALrefold•soluble•protein•ref.•column•(large)#10                                 88
DUALmembrane•starter•kit•SUC•+•Mouse•whole•embryo,•11•days•cDNA•library•(NubG-x)     27   DUALrefold•soluble•protein•ref.•column•(large)#2                                  88
DUALmembrane•starter•kit•SUC•+•Normalized•human•fetal•brain•cDNA•library••(NubG-x)   27   DUALrefold•soluble•protein•ref.•column•(large)#3                                  88
DUALmembrane•starter•kit•SUC•+•Normalized•human•spleen•cDNA•library•(NubG-x)         27   DUALrefold•soluble•protein•ref.•column•(large)#4                                  88
DUALmembrane•starter•kit•SUC•+•Normalized•mouse•brain•cDNA•library•(NubG-x)          27   DUALrefold•soluble•protein•ref.•column•(large)#5                                  88
DUALmembrane•starter•kit•SUC•+•Normalized•mouse•embryo•cDNA•library•(x-NubG)         27   DUALrefold•soluble•protein•ref.•column•(large)#6                                  88
DUALmembrane•starter•kit•SUC•+•Rat•neonatal•cardiomyocyte•cDNA•library•(NubG-x)      27   DUALrefold•soluble•protein•ref.•column•(large)#7                                  88
DUALmembrane•starter•kit•SUC•+•S. cerevisiae•cDNA•library•(NubG-x)                   27   DUALrefold•soluble•protein•ref.•column•(large)#8                                  88
DUALmembrane•vector•complementation•set•N                                            34   DUALrefold•soluble•protein•ref.•column•(large)#9                                  88
DUALmembrane•vector•complementation•set•STE                                          35   DUALrefold•soluble•protein•ref.•column•(small)•#1                                 88
DUALmembrane•vector•complementation•set•SUC                                          35   DUALrefold•soluble•protein•ref.•column•(small)•#10                                88

                                                                                                                                                                                 101
12      Alphabetical product listing


      DUALrefold•soluble•protein•ref.•column•(small)•#2   88   DUALXtract•cytoplasmic•and•nuclear•protein•extraction•kit     85
      DUALrefold•soluble•protein•ref.•column•(small)•#3   88   DUALXtract•mammalian•cell•total•protein•extraction•reagent    82
      DUALrefold•soluble•protein•ref.•column•(small)•#4   88   DUALXtract•total•membrane•protein•extraction•kit              83
      DUALrefold•soluble•protein•ref.•column•(small)•#5   88
                                                               -E-
      DUALrefold•soluble•protein•ref.•column•(small)•#6   88
                                                               E.coli REG-12•expression•strain                               70
      DUALrefold•soluble•protein•ref.•column•(small)•#7   88
                                                               EasyClone•cDNA•library•construction•kit                       63
      DUALrefold•soluble•protein•ref.•column•(small)•#8   88
                                                               EasyClone•normalized•cDNA•library•construction•package        63
      DUALrefold•soluble•protein•ref.•column•(small)•#9   88
                                                               EasyClone•PCR•kit                                             64
      DUALrefold•soluble•protein•refolding•kit            88
                                                               ECS•affinity•resin                                            74
      dualTRAP•affinity•resin,•250•ml                     70
                                                               -F-
      dualTRAP•affinity•resin,•30•ml                      70
                                                               FlexIP•immunoprecipitation•kit                                66
      dualTRAP•multiwell•affinity•plates,•1•pc            70
      dualTRAP•multiwell•affinity•plates,•5•pcs           70   -H-
      dualTRAP•spin•columns,•10•pcs                       70   HeLa•cell•line•cDNA•library•(NubG-x)                          36
      dualTRAP•spin•columns,•100•pcs                      70   HeLa•cell•line•cDNA•library•(pGAD-DS)                         15
      dualTRAP•spin•columns,•25•pcs                       70   HTX•beta-galactosidase•assay•kit                              50
      DUALXpress•ELUTION•buffer,•20•ml                    70   Human•adult•brain•cDNA•library•(NubG-x)                       36
      DUALXpress•INDUCER•solution,•20•ml                  70   Human•adult•brain•cDNA•library•(x-NubG)                       36
      DUALXpress•starter•kit                              70   Human•adult•colon•cDNA•library•(NubG-x)                       36
      DUALXtract•buffer•1                                 84   Human•adult•colon•cDNA•library•(pGAD-DS)                      15
      DUALXtract•buffer•10                                84   Human•adult•kidney•cDNA•library•(NubG-x)                      36
      DUALXtract•buffer•11                                84   Human•adult•kidney•cDNA•library•(x-NubG)                      36
      DUALXtract•buffer•12                                84   Human•adult•liver•cDNA•library•(NubG-x)                       36
      DUALXtract•buffer•2                                 84   Human•adult•lung•cDNA•library•(NubG-x)                        36
      DUALXtract•buffer•3                                 84   Human•embryonal•brain•cDNA•library•(NubG-x)                   36
      DUALXtract•buffer•4                                 84   Human•hepatocellular•carcinoma•cells•cDNA•library•(pGAD-DS)   15
      DUALXtract•buffer•5                                 84
                                                               -J-
      DUALXtract•buffer•6                                 84
                                                               Jurkat•T•cDNA•library•(NubG-x)                                36
      DUALXtract•buffer•7                                 84
      DUALXtract•buffer•8                                 84   -K-
      DUALXtract•buffer•9                                 84   KickStart•protein•expression•kit                              73
      DUALXtract•buffer•set                               84   -L-
      DUALXtract•buffer•set•(trial•size)                  84   LNCaP•cDNA•library•(pGAD-DS)                                  15

102
                                                                                               Alphabetical product listing         12


LNCaP•cells•cDNA•library•(NubG-x)                     36   -P-
Lytic•Agent•for•384•preps                             52
                                                           p413CYC•yeast•expression•vector                                    76
Lytic•Agent•for•3840•preps                            52
                                                           p413GAL-L•yeast•expression•vector                                  76
Lytic•Agent•for•96•preps                              52
                                                           p413GAL-S•yeast•expression•vector                                  76
-M-                                                        p414CYC•yeast•expression•vector                                    76
Mammary•epithelial•cells•cDNA•library•(NubG-x)        36   p414GAL-L•yeast•expression•vector                                  76
Media•package•1                                       54   p414GAL-S•yeast•expression•vector                                  76
Media•package•2                                       54   p414TEF•yeast•expression•vector                                    76
Medicago•nodules•cDNA•library•(NubG-x)                36   p415ADH•yeast•expression•vector                                    76
Medicago•nodules•cDNA•library•(x-NubG)                36   p415CYC•yeast•expression•vector                                    76
Mouse•adult•whole•brain•cDNA•library•(NubG-x)         36   p415GAL-L•yeast•expression•vector                                  76
Mouse•adult•heart•cDNA•library•(NubG-x)               36   p415GAL-S•yeast•expression•vector                                  76
Mouse•adult•heart•cDNA•library•(pGAD-DS)              15   p416CYC•yeast•expression•vector                                    76
Mouse•adult•heart•cDNA•library•(x-NubG)               36   p416GAL-L•yeast•expression•vector                                  76
Mouse•adult•kidney•cDNA•library•(NubG-x)              36   p416GAL-S•yeast•expression•vector                                  76
Mouse•adult•spleen•cDNA•library•(x-NubG)              36   p417-CYC•yeast•expression•vector                                   75
Mouse•anti•c-myc•antibody                             53   p423CYC•yeast•expression•vector                                    76
Mouse•anti•HA•antibody                                53   p423GAL-L•yeast•expression•vector                                  76
Mouse•anti•LexA•antibody                              53   p423GAL-S•yeast•expression•vector                                  76
Mouse•embryo•11d•cDNA•library•(pGAD-DS)               15   p424ADH•yeast•expression•vector                                    76
Mouse•whole•embryo,•11•days•cDNA•library•(NubG-x)     36   p424CYC•yeast•expression•vector                                    76
NMY51•yeast•reporter•strain                           47   p424TEF•yeast•expression•vector                                    76
NMY61•yeast•reporter•strain                           47   p425ADH•yeast•expression•vector                                    76
NMY70•yeast•reporter•strain                           47   p425CYC•yeast•expression•vector                                    76
-N-                                                        p425GAL-L•yeast•expression•vector                                  76
                                                           p425GAL-S•yeast•expression•vector                                  76
Normalized•human•fetal•brain•cDNA•library••(NubG-x)   36
                                                           p425TEF•yeast•expression•vector                                    76
Normalized•human•spleen•cDNA•library•(NubG-x)         36
                                                           p426CYC•yeast•expression•vector                                    76
Normalized•mouse•brain•cDNA•library•(NubG-x)          36
                                                           p426GAL-L•yeast•expression•vector                                  76
Normalized•mouse•embryo•cDNA•library•(x-NubG)         36
                                                           p426GAL-S•yeast•expression•vector                                  76
Normalized•human•fetal•brain•cDNA•library•(pGAD-DS)   15
                                                           p426TEF•yeast•expression•vector                                    76
Normalized•mouse•brain•cDNA•library•(pGAD-DS)         15
                                                           p427-TEF•yeast•expression•vector                                   75
                                                           pALEX2a•DUALXpress•vector                                          70
                                                                                                                                   103
12     Alphabetical product listing


      pALEX2b•DUALXpress•vector                      70   pVB1•expression•vector                                            72
      pALEX2a,b,c•DUALXpress•vector•set              70   pVB2•expression•vector                                            72
      pALEX2c•DUALXpress•vector                      70   pVB3•expression•vector                                            72
      pALEX2-CT•DUALXpress•vector                    70   pVB4•expression•vector                                            72
      PARP-1•assay•kit                               68   pVB5•expression•vector                                            72
      pBT3-C•DUALmembrane••bait•vector               32   pVB6•expression•vector                                            72
      pBT3-N•DUALmembrane•bait•vector                32   pVB7•expression•vector                                            72
      pBT3-STE•DUALmembrane••bait•vector             32   pVB8•expression•vector                                            72
      pBT3-SUC•DUALmembrane••bait•vector             32   pVB9•expression•vector                                            72
      pDHB1•DUALhunter•bait•vector                   45   pY3H•DUALhybrid•bridge•vector                                     14
      pFL-MEX•mammalian•expression•vector            81   -Q-
      pGAD-HA•DUALhybrid••prey•vector                14
                                                          QuickLyse•pEAS1-a•expression•vector                               71
      pGAL-MF•yeast•expression•vector                75
                                                          QuickSpin•detergent•removal•kit                                   92
      pHA-MEX•mammalian•expression•vector            81
                                                          QuickSpin•protein•concentration/•buffer•exchange/•desalting•kit   91
      pKS1•tagless•expression•vector                 74
      pKS1-ST•Strep-tag•expression•vector            74
                                                          -R-
      pKS1-ST-Gal•control•vector                     74   Rabbit•polyclonal•anti•ECS•antibody                               53
      pKS2-ST•Strep-tag•secreted•expression•vector   74   Rabbit•polyclonal•anti•LYTAG•antibody                             53
      pKS3-FL•ECS-tag•expression•vector              74   Rat•neonatal•cardiomyocyte•cDNA•library•(NubG-x)                  36
      pKS4-FL•ECS-tag•secreted•expression•vector     74   -S-
      pLexA-C•DUALhybrid•bait•vector                 14   S. cerevisiae•cDNA•library•(NubG-x)                               36
      pLexA-N•DUALhybrid••bait•vector                14   S. cerevisiae•cDNA•library•(pGAD-DS)                              15
      pMEX-FL•mammalian•expression•vector            81   Single-stranded•carrier•DNA                                       49
      pMEX-HA•mammalian•expression•vector            81   SpeedPrep•yeast•plasmid•isolation•kit•                            51
      pPR3-C•DUALmembrane•prey•vector                32   Stable•cell•line•generation•part•I                                79
      pPR3-N•DUALhunter•prey•vector                  45   Stable•cell•line•generation•part•II                               79
      pPR3-N•DUALmembrane•prey•vector                32
                                                          -T-
      pPR3-STE•DUALmembrane•prey•vector              32
                                                          TOTAL•protein•assay•reagent                                       87
      pPR3-SUC•DUALmembrane•prey•vector              32
                                                          TOTAL•protein•extraction•kit                                      86
      PROLYSE•extraction•solution,•100•ml            70
                                                          -U-
      PROLYSE•extraction•solution,•25•ml             70
                                                          Universal•kinase•assay•kit                                        67
      Protein•stability•and•aggregation•assay•kit    90
      pTEF-MF•yeast•expression•vector                75

104
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USA and Canada             Europe                Asia                            Asia
Dualsystems Biotech Inc.   Slovakia              China                           Singapore
PO Box 90734               Biohem                Dakewe Biotech                  Sciencewerke
San Pedro, CA 90734        www.biohem.sk         www.dakewe.com                  www.sciencewerke.com
Phone +1 650 678 53 24     biohem@biohem.sk      order@dakewe.com                enquiry@sciencewerke.com
Fax +1 866 434 99 33
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Europe                     www.ecogen.com        www.viswagenbiotech.com         www.bioclone.co.kr
Germany                    ecogen@ecogen.com     customer.service@viswagen.com   bioclone@bioclone.co.kr
Biozol GmbH
www.biozol.de              United Kingdom,       Joyvel Bioscience               Taiwan
info@biozol.de             Republic of Ireland   www.joyvel.com                  GenDiscovery Biotechnology
                           Stratech Scientific   joyvelbio@gmail.com             www.gendiscovery.com.tw
France                     www.stratech.co.uk                                    gene@gendiscovery.com.tw
Euromedex                  info@stratech.co.uk   Biolinkk
www.euromedex.com                                www.biolinkk.com                If no distributor is listed for your
order@euromedex.com        Middle East           biolinkk@gmail.com              country, please contact us directly
                           Israel                                                Dualsystems Biotech AG
Netherlands,               Almog Diagnostics     Japan                           Grabenstrasse 11a
Belgium and                www.almog.co.il       Cosmo Bio                       8952 Schlieren
Luxemburg                  info@almog.co.il      www.cosmobio.co.jp              Switzerland
DivBio Science                                   mail@cosmobio.co.jp             Phone +41 44 738 5000
www.divbio.nl                                                                    Fax +41 44 738 5005
info@divbio.nl                                                                   info@dualsystems.com
                                                                                 www.dualsystems.com
                      Dualsystems Biotech Inc.   Dualsystems Biotech AG
                      P O Box 6698               Grabenstrasse 11a
                      San Pedro, CA 90734/USA    CH-8952 Schlieren/Switzerland

                      Phone +1 650 678 53 24     Phone +41 44 738 50 00
                      Fax   +1 866 434 99 33     Fax   +41 44 738 50 05

www.dualsystems.com   info_us@dualsystems.com    info@dualsystems.com

								
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