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MP-DFA94

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									Manual

Omscientia Memb-PASSTM Differential
Filtration Detergent Assay (DFDA)

Cat. No.                        Amount
                                                                               Introduction
MP-DFA94                        94 solutions, 150 μl each
                                                                               The Omscientia Memb-PASS™ Differential Filtration
                                                                               Detergent Assay is a simple method for assessing the
For in vitro use only                                                          stability of a membrane protein in a panel of 94
Store at 4°C                                                                   detergents.

Omscientia       Memb-PASS™                        Differential                The protein is bound to an affinity resin, washed
Filtration Detergent Assay                                                     extensively with buffer containing the new detergent
                                                                               and finally eluted in that new detergent containing
•    94 detergent panel provided in a 96 deep-well
                                                                               buffer. Protein that is not stable in the new detergent is
     block, 150 μl at 2x working concentration each; see
                                                                               precipitated on the resin and therefore not present in
     data sheet for screen formulation
                                                                               the eluates.

Materials Needed                                                               Half of each respective eluate is passed through a
                                                                               high MWCO filter microplate and the other half
•    Omscientia Memb-PASS™ high and low MWCO
                                                                               through a low MWCO filter microplate.
     filtration plates (Cat.# MP-MWCO-Kit)
                                                                               The elutions from the plates are quantified using a dot-
•    0.2 μm filter microplate (e.g. AcroPrep™ 96-well
                                                                               blot and Western detection method. The high MWCO
     Filter Plates)
                                                                               values provide readout on stability while the ratio of
•    PCR plates and appropriate plate adapter                                  low    MWCO/high         MWCO        provides     sizing
•    Elution plates and appropriate plate adapter                              information.

•    Currently used detergents and buffers
                                                                               Prerequisites
•    2x wash buffer
                                                                               The protein must not be proteolysed.
•    2x elution buffer                                                         If the protein is proteolysed, it must be purified prior to
•    Affinity resin                                                            performing the assay since the detection method
                                                                               allows no distinction between proteolysed and non-
•    Dot-blot apparatus                                                        proteolysed protein.
•    Microplate holders for centrifuge
•    IR-dye conjugated antibody to protein or tag
•    Fluorescence plate reader/imager




                  Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
Page 1 of 2                                               http://www.jenabioscience.com                                       Last update: Jan 19, 2012
Manual

Omscientia Memb-PASSTM Differential
Filtration Detergent Assay (DFDA)
Protocol                                                                       Detergent Exchange
Check the detergent stock plate. If necessary,                                 13. Add 30 μl of the prepared wash buffer (step #4)
resuspend the detergents by gentle warming.                                        to each well, spin the plate at 2000g for 2 min
                                                                                   at 4°C and discard the flow-through.
Add positive and negative control
                                                                               14. Repeat the last step 5 more times to exchange the
The 94 detergent panel in the 96 deep-well block provides two                      detergent. On the last addition of wash buffer
empty positions for a negative (A1) and positive (A2) control,
                                                                                   you can use the rest instead of 30 μl only.
respectively.
                                                                               15. Place the 0.2 μm filter microplate containing the
1.     Add 150 μl ultrapure water to A1 (negative                                  bound protein onto a new 96 well elution plate.
       control) and 150 μl of 2x current detergent to A2                       16. Add 70 μl of the prepared elution buffer (step #2
       (positive control) of the provided deep-well block                          and #3) to each well and spin the plate at
       containing the 94 detergent panel.                                          2000g for 2 min at 4°C, keep the flow-through.
Preparing the Elution Buffer Plate                                             Differential Filtration
2.     Dispense 40 μl of 2x elution buffer to each well                        17. Assemble two more elution plates with an
       of 96 well PCR plate.                                                       Omscientia Memb-PASS™ high and low MWCO
3.     Add 40 μl of the detergent stock plate and mix                              filtration plate, respectively.
       carefully by slowly pipetting up and down                               18. Add 30 μl of the elution from step #16 to each of
       several times.                                                              the MWCO filtration plates.
Preparing the Wash Buffer Plate                                                19. Spin both plates at 2000g for 4 min at 4°C,
                                                                                   keep the flow-through. If the elution volumes of
4.     Add 110 μl of 2x wash buffer to each well of the                            the low MWCO plate seem low, spin again for
       remaining detergent stock plate and mix carefully                           another 2 min.
       by slowly pipetting up and down several times.
                                                                               Protein Analysis
Protein Binding
                                                                               Apply e.g. Dot Blot and follow the instrument instructions.
5.     Chose a suitable resin (e.g. Ni-NTA, Co-TALON)
                                                                               20. Quantify the protein amounts in each well of both
       and equilibrate the necessary amount for 500
                                                                                   elution plates.
       μg. Make sure that the binding capacity of your
                                                                               21. Normalize the values to the highest intensity dot
       resin allows complete protein binding.
                                                                                   on each respective blot.
6.     Add ~500 μg of protein.
                                                                               22. Display the following results:
7.     Add buffer to obtain a 20% slurry (e.g. 1.5 ml
                                                                                   o High MWCO (provides information on
       resin + 6 ml buffer).
                                                                                       protein stability)
8.     Batch bind overnight at 4°C.
                                                                                   o Low       MWCO/High          MWCO       (provides
Protein Washing                                                                        information on relative PDC size)
                                                                               23. Perform further analysis (e.g. gel filtration) with
9.  Place the 0.2 μm filter microplate onto a new 96
                                                                                   the most promising detergents, if desired.
    well elution plate.
10. Pipette 50 μl of the protein bound slurry into each
    well of the 0.2 μm filter microplate using a                               References
    multichannel pipette (tip ends cut).                                       [1] Vergis et al. (2010) A high-throughput differential filtration
                                                                               assay to screen and select detergents for membrane proteins. Anal.
11. Add ~150 μl of current detergent buffer to each
                                                                               Biochem. 407:1.
    well of the filter microplate, spin the plate at
                                                                               [2] Vergis et al. (2011) The variable detergent sensitivity of
    2000g for 2 min at 4°C and discard the flow-
                                                                               proteases that are utilized for recombinant protein affinity tag
    through.                                                                   removal. Prot. Expr. Purif. 78:139.
12. Repeat the last step 3 more times to remove
    unbound protein.


                  Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
Page 2 of 2                                               http://www.jenabioscience.com                                       Last update: Jan 19, 2012

								
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