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Protein MicroArray Program

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Protein MicroArray Program
800 Research Parkway                                       a 3-dimensional substrate for protein
Meriden, CT 06450 U.S.A.                                          microarray applications
Tel: (203) 639-2598
Toll Free: (800) 323-1891                                          (for research use only)
Customer Support: (800) 445-7426
Fax: (617) 426-3908                                                 NOT FOR RESALE
Web site: http://www.perkinelmer.com
Email: hydrogel@packardbioscience.com

PerkinElmer Life Sciences International Offices:                     Protocol Guide
Australia +(61)-3-95434266; Austria +(43)-1-2702504;
Belgium +(32)-2-4818530;
Canada (Main Office) +(1)-905-673-8028;
Central Europe +(43)-2-23037000;
Denmark +(45)-43-909023; France +(33)-1-46862775;
Germany +(49) 6103 385151; Italy +(39)-02-33910796;
Japan +(81)-3-38665850; Netherlands +(31)-50-5445900;
Pacific Rim +(852) 2620-1881; Russia +(7095)-4296577;
                                                                 www.perkinelmer.com
Switzerland +(41)-1-4816944;
United Kingdom +(44)-118-9844981

For all other international representatives, contact the
PerkinElmer Life Sciences U.S.A. office.
Thank you for choosing HydroGelTM coated slides for            Nomenclature
your microarray needs!                                         In the microarray literature there exist at least two nomenclature
                                                               systems for referring to binding partners. Both use common terms:
                                                               “probes” and “targets”. We use the following terminology.
Table of Contents
Product Description __________________________ 2               “Probe” is the biological sample fixed to the solid support. For protein
                                                               arrays this refers to proteins that are regularly arranged on a solid
Contents of Package _________________________ 3                substrate and can bind to the target proteins to be detected. For
                                                               instance, the probe protein may be selected from the group
Additional Materials and Equipment Required_____ 3             consisting of antigens, antibodies, receptors, enzymes, etc., with
                                                               antibodies representing a class of the most commonly used probes.
Storage and Handling Prior to Use ______________ 4
Product Use Guidelines _______________________ 4               “Immobilization” is the process of fixing/binding the probe to the
                                                               substrate, in this case the HydroGel coated slide.
  Preparing HydroGel Coated Slides for Printing _______5
  Printing Buffers and Conditions ___________________6         “Target” is the solution phase biological molecule that will bind to the
                                                               probe. Targets are usually derived from a biological sample to be
  Immobilization of Proteins on HydroGel Coated Slides 6       analyzed. “Targets” can refer to a protein, nucleic acid, or other
                                                               molecule that is included in samples to be tested and can bind the
  Blocking _______________________________________7            probe proteins immobilized on the protein chip.
  Assays and Washes _____________________________7
                                                               “Source Plate/Sample Source” is the microtiter plate filled with
  Imaging________________________________________8             probes to be dispensed. Both 96 and 384 well microplates are
                                                               commonly used formats.
Troubleshooting Guide _______________________ 9
Nomenclature ______________________________ 10                  “Array” is an ordered (often orthogonal) set of spots obtained by
                                                               dispensing of biological samples (probes) onto a substrate, in this
                                                               case, a HydroGel coated slide. One array may contain one or
                                                               multiple replicates of the same probe.




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Troubleshooting Guide                                                                        Product Description
   Symptom            Potential Cause                              Solution                                         TM
                                                                                             PerkinElmer HydroGel coated slides provide a 3-dimensional
Damaged, ripped, or     Contact with substrate when in   Avoid contact with the
missing substrate       hydrated form                    HydroGel substrate                  substrate for a variety of protein microarray applications. The
                                                                                                                                       TM
                        Large osmotic changes            Avoid switching HydroGel            specialized formulation of the HydroGel substrate provides a
                                                         coated slides between high          combination of low inherent fluorescence, low non-specific binding,
                                                         and low osmotic strength            and high probe loading capacity while maintaining protein
                                                         solutions
Diffuse or very large   HydroGel coated slides were      Carefully follow the procedure
                                                                                             functionality and accessibility. This unique combination of physical
spots                   not washed prior to printing     listed in “Preparing HydroGel       and chemical properties allows for the development of sensitive,
                                                         coated slides for printing”         reproducible assays having a broad dynamic range. HydroGel
                        Incompletely dried substrate     Carefully follow the procedure      coated slides are manufactured and packaged in our Class 1000
                        prior to printing                listed in “Preparing HydroGel
                                                                                             clean room to minimize dust and other debris that can interfere with
                                                         coated slides for printing”
Holes in center of      Excessive force applied to       Optimize printer overtravel         assays.
printed spots           substrate by contact printer     and/or substrate thickness
                                                         settings                                    12 X 12 HydroGel™ Coated Slide
                        Storage of unprinted slides      Do not rinse storage agent
                        after water rinses               from HydroGel coated slides




                                                                                                 AAA00
                                                         until immediately before use




                                                                                                    t
                                                                                                             ABC
Weak assay signal       Low probe immobilization         Extend immobilization time;




                                                                                                  000




                                                                                                                                                 12mm
                                                         carry out immobilization in a
                                                         chamber containing a dish of
                                                         saturated sodium chloride
                                                         solution; test alternate printing
                                                         buffers                                                                        12mm
                        Insufficient target incubation   Extend target incubation time                    23mm                 15mm              13mm
                        time
                        Poorly labeled target            Check efficiency of target                  12 X 40 HydroGel™ Coated Slide
                                                         labeling
                        Degraded target                  Check quality of sample
                                                         preparation (via gel
                                                                                                                               40mm




                                                                                                  AAA0
                                                         electrophoresis for example)




                                                                                                 t0000

                                                                                                  ABC
                        Insufficient mixing during       Agitate slides on a rocking




                                                                                                                                                        12mm
                        target incubation                platform shaker during target
                                                         sample incubation
High Background         Labeled protein level too high   Use a more dilute target
                                                         sample; include a pre-blocking
                                                         step; extend washing time
                        Drying during target sample      Place slides in a humidified                    22.2mm                                  12.8mm
                        incubation                       environment to reduce
                                                         evaporation from under cover        HydroGel coated slides are dried prior to printing. In this state, the
                                                         glasses                             HydroGel substrate supports both non-contact and contact printing.
                                                                                             Contact printers may require an initial adjustment of the over-travel
                                                                                             or substrate thickness settings to produce high quality arrays.
                                                                                             Guidelines for this process are included on a separate sheet.

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Immobilization of proteins within the HydroGel substrate requires no          The concentration of target, the sample/reaction volume needed for
protein modifications. Proteins are irreversibly immobilized within the       the assay, the assay temperature and the incubation times are assay
porous HydroGel substrate through interactions with the matrix. The           dependent. In general, good performance on HydroGel coated slides
3–dimensional nature of the substrate is thought to minimize protein          is achieved using sample incubation times from 30 minutes to two
denaturation during this process.                                             hours. Depending on the size of the target, its concentration, and the
                                                                              nature of the probes, incubation steps may need to be extended (up
                                                                              to overnight is possible).
Contents of Package
25 or 100 HydroGel coated slides                                              For dilution of samples, we recommend the use of PBST, however
HydroGel Coated Slide Protocol Guide                                          both the sample dilution buffer and the wash buffer can be varied
Contact Printer Adjustment Guide                                              based on the assay requirements. Samples containing a purified
                                                                              target should be supplemented with 1% BSA. If other buffering
Additional Materials and Equipment Required                                   systems are used, rapid changes in osmotic strength should be
Printing buffer options:                                                      avoided, as this may lead to substrate damage.
0.1M sodium phosphate buffer (pH 7.2)
1X PBS [0.01M sodium phosphate (pH 7.4), 0.14M sodium chloride,               Agitation of the HydroGel coated slides on a rocking platform shaker
0.003M potassium chloride]                                                    during incubations yields optimal performance and sensitivity.
0.05M borate buffer (pH 9.0)
Other:                                                                        Owing to the 3-dimensional nature of the HydroGel substrate, slightly
PBST (1X PBS, 0.5% Tween 20)- sample dilution and wash buffer                 longer washes may be required than those typically used on 2-
Bovine Serum Albumin- blocking agent                                          dimensional substrates. Post-target incubation wash conditions are
(Optional) Fluorescently labeled protein- indicator for printer               as follows:
adjustment
Equipment:                                                                       Rinse briefly (for 20 seconds) in PBST (PBS + 0.5% Tween
Printer- for example:    PerkinElmer BioChip Arrayer (non-contact)               20), followed by three 10 minute washes in fresh changes of
                                                    TM                            PBST with gentle agitation. Depending on the sample and
                         or PerkinElmer SpotArray 24 (contact)
Scanner- for example: PerkinElmer ScanArray series confocal                      detection method, the washes may need to be extended to as
                         laser scanner                                            long as 20 minutes each (e.g. when using a directly labeled
Incubator/oven                                                                    complex protein sample containing a high fluorophore
Platform Shaker                                                                   concentration). Slides exposed to different target samples should
                                                                                  be processed separately.

                                                                              Imaging
                                                                              HydroGel coated slides can be imaged in any standard microarray
                                                                              slide scanner, for example PerkinElmer ScanArray. HydroGel
                                                                              coated slides should be imaged dry.




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Blocking                                                                   Storage and Handling Prior to Use
Although the HydroGel substrate inherently has very low associated         HydroGel coated slides are stable for many months when stored at
non-specific binding, blocking has been shown to improve the               room temperature inside the sealed shipper. Refrigeration or
resolution of low-abundance targets and consequently may improve           desiccation of HydroGel coated slides is not recommended. After
the detection limit of some assays. We recommend that a pre-               opening, unused slides should be placed in the original slide box and
blocking step be included prior to the addition of target.                 the slide box returned to the resealable shipper. Do not store used
                                                                           HydroGel coated slides with unused HydroGel coated slides.
1. Block in PBST + 1%BSA for one hour at room temp with
   gentle agitation.                                                       The preprinting wash procedure described below is designed to
2. Briefly rinse three times with PBST.                                    remove a storage agent present in the HydroGel substrate. After
                                                                           washing, do not return unprinted HydroGel coated slides to dry
Assays and Washes                                                          storage, as they may be susceptible to damage during the printing
There are several methods for setting up assays/hybridizations on          and probe immobilization processes. Note that with two-pad
HydroGel coated slides. The most convenient way is by use of               HydroGel coated slides, both pads must be used immediately
disposable gasket-style hybridization chambers. Grace BioLabs sells        following pre-print washing. One pad cannot be used while the other
           TM
HybriWells (catalog numbers HBW75 or HBW2240;                              is “stored” as this can result in damage to the second substrate pad.
www.gracebio.com) that fit the single pad HydroGel coated slides.
MJ Research sells Frame-Seal™ incubation chambers (catalog                 HydroGel coated slides should be used in a dust free, clean
numbers SLF-0601; http://www.mjresearch.com/) that fit the two-pad         environment and care should be taken to avoid contact with the
HydroGel coated slides. When using these chambers, dry the                 coated portion of the slides. Do not use colored ink for labeling
HydroGel coated slides as described above, apply the adhesive              HydroGel coated slides as this may contribute to background
gaskets and then the appropriate amount of sample depending on             fluorescence.
which gaskets are being used (detailed directions for use are
supplied by the manufacturer). Alternately, approximately 50 to 100
μL of sample can be applied to the array, followed by sealing with a
                                                                           Product Use Guidelines
22 X 50 mm cover glass. When using the cover glass method, the             The pre-printing and probe immobilization protocols below should be
slides should be placed in a humidified chamber to prevent                 followed carefully to ensure optimal performance of the HydroGel
evaporation.                                                               substrate. HydroGel coated slides support a number of different
                                                                           protein microarray applications. Therefore, assay protocols are
                                                                           largely assay dependent and need to be determined by the
                                                                           investigator. In general, assay protocols designed for use on 2-
                                                                           dimensional substrates can be used on HydroGel coated slides with
                                                                           some minor modifications; these are discussed below.




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                                                                        Printing Buffers and Conditions
Preparing HydroGel Coated Slides for Printing                           The HydroGel substrate is compatible with phosphate and borate
The purpose of the following pre-printing steps is to remove a          buffer systems and glycerol concentrations up to 40%. The HydroGel
storage agent present in the HydroGel substrate and to ensure a         substrate is stable from pH 5 to 9. Protein immobilization is affected
consistent, uniform substrate condition prior to printing.              by pH.

1. Briefly rinse HydroGel slides three times in distilled water         To avoid edge effects, do not print within 1 mm from the edges of the
   (dH2O), followed by three washes in dH2O for 10 minutes              HydroGel substrate.
   each, and finally six brief rinses in dH2O. All wash steps are
   most effective when carried out in a large volume, for example,      Immobilization of Proteins on HydroGel Coated
   by grouping the slides in a 10 slide-capacity glass slide dish and   Slides
   washing with a 200mL volume. Always use fresh changes of             The immobilization of proteins within the HydroGel substrate
   dH2O for each wash step.                                             correlates directly with post-printing incubation time and is
2. Remove excess/free water. Excess water can be removed by             dependent on both the printing buffer and the proteins themselves.
   spinning the HydroGel coated slides gently in a benchtop
   centrifuge (at approximately 1000 to 1500 RCF for 5 minutes).        1. Incubate arrays in a humidified chamber. Efficient
   Caution: when hydrated, the HydroGel substrate is not rigid and         immobilization requires an extended post-printing incubation (8
   centrifuging at higher speeds or for longer times may damage            to 16 hours). For thermally stable proteins the incubation should
   the coating. Alternately, HydroGel coated slides can be dried by
                                                                           be conducted at 30C. Placing a saturated sodium chloride
   placing them vertically on top of an absorbent surface for several
                                                                           solution at the bottom of a standard incubator at 30 C yields the
   minutes to allow excess water to run off the slides. Caution: do
                                                                           proper level of relative humidity (approximately 65%). When
   not place HydroGel coated slides substrate side down or directly
                                                                           using a particularly fragile, thermal sensitive, or impure protein
   blot the HydroGel substrate, as this will cause damage. When
                                                                           the incubation can be carried out at a lower temperature, such as
   dry, the HydroGel substrate has a clear, smooth and thin
   appearance.                                                             4C. Note however that the incubation time will have to be
                                    o
3. Place HydroGel slides in a 40 C incubator/oven for 20                   extended when the incubation is carried out at a lower
   minutes. Caution: printing on wet slides can sometimes lead to          temperature. If data are to be quantitatively compared among
   substrate damage and undesirably large, poorly shaped spots.            multiple days, this incubation time should be held constant.
4. Remove from incubator/oven and allow slides to cool to               2. Rinse briefly (for 20 seconds) in PBST (PBS + 0.5% Tween
   room temperature (approximately 5 minutes).                             20), followed by three 30 minute washes with PBST.
                                                                        3. (Optional) Dry HydroGel coated slides. After printing and
                                                                           immobilization, slides can be stored in blocking solution (with the
                                                                           addition of 0.01% sodium azide) or dry at 4C (non-condensing
                                                                           atmosphere). No significant loss of probe activity (either IgG or
                                                                           streptavidin probes) was found for up to one week of storage in
                                                                           either condition. Since the stability of printed probes is protein
                                                                           dependent, the user is encouraged to evaluate the storage
                                                                           periods for the specific assay.


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