Memb-PASS

Omscientia, Inc.
Memb-PASS Detergent Stability Assay Detailed Protocol
Protocol Updated 6/22/11 by Rachel Reuther Michael Wiener, James Vergis and Craig Sterling Omscientia, Inc., PO Box 523, Mammoth Lakes, CA 93546 Memb-PASS is patent-pending and available exclusively from Omscientia, Inc. 2010 Omscientia, Inc.

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Background
The Omscientia Memb-PASS Differential Filtration Detergent Assay from Omscientia, Inc. is a simple method for assessing the stability of a membrane protein in a panel of 94 detergents. The protein is bound to an affinity resin, washed with the new detergent, eluted in the new detergent, and the eluate is passed through two different Omscientia MWCO filterplates to obtain stability and relative size information. The elutions from the microplates are quantified using a dot-blot and and Western detection method and an Infrared Imager using infrared fluorescence detection for Western Blot, protein detection, Western blot Analysis. More detection methods are being developed by Omscientia and will be released in the near future.

Materials Needed
Affinity resin Omscientia Differential Filtration Detergent Assay (150l) #MP-DFA94 2X wash buffer 2x elution buffer Filtered microplates for holding resin o Pall AcroPrep 96 0.2m GHP plates High and Low MWCO plates for sizing o Omscientia High MWCO #MP-MWCO-5-H or MP-MWCO-25-H o Omscientia Low MWCO #MP-MWCO-5-L or MP-MWCO-25-L o Omscientia High/Low MWCO combo pack #MP-MWCO-Kit Waste collection plates (for washes) o CoStar 3631 Flat bottom 96 well plates Eluate collection plates o Fisher AB1127 0.2ml V-bottom storage plates PCR collection plate adapters o Pall #5226 Dot-blot apparatus Microplate holders for centrifuge IR-dye conjugated Ab to protein or tag Fluorescence Plate Reader/Imager (i.e. LI-COR Odyssey)

Prerequisites
Protein of interest must be non-proteolysed. If the protein expresses as the full length construct then the protein does not have to be purified prior to performing the assay. If the protein is proteolysed, then it must be purified prior to performing the assay since there is no separation of full length from cut protein and thus both would light up on the Western dot-bot.

Memb-PASS is patent-pending and available exclusively from Omscientia, Inc. 2010 Omscientia, Inc.

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Protein of interest must be extracted from the membrane in a good detergent. While there is evidence of some rescue, starting with a bad detergent is not recommended. This will most likely involve performing a gel-filtration run of purified protein to assess that detergent.

Protocol
1. Thaw detergent stock plate (depending on how stored) a. You may need to re-suspend one detergent by gentle heating 2. Make Wash and Elution buffers a. Dispense 40l of 2X Elution buffer into a 96 well PCR plate b. Add 40l of 2X detergent stock to elution plate, mixing the solutions by pipetting up and down several times c. Add 110l of 2X Wash buffer to the remaining 110l of 2X detergent stock plate, mixing the solutions by pipetting up and down several times. 3. Bind protein to resin = you can either batch bind to resin and then dispense the protein-bound resin into the plate or dispense resin into the wells 1st and then add the protein. a. Batch Binding Method: i. Add 2.4ml of 50% Talon slurry to a 15mL tube, and equilibrate resin ii. Add 500ug of protein and bring the total volume up to 6mL iii. Batch bind to resin O/N at 4C iv. Cut the tips off 200l tips to create a wider bore v. Pipette 50l of slurry into each well of a 96-well filter plate (0.2m filter) b. Plate Binding Method: i. Cut the tips off 20l tips to create a wider bore ii. Pipette 20l of 50% resin into each well of the microplate iii. Add water to each well and spin at 2000xg for 2min to remove EtOH iv. Add buffer to each well and spin at 2000xg for 2min to equilibrate resin v. Add protein to each well and let incubate in microplate shaker vi. Spin at 2000xg for 2min to remove unbound protein 4. Wash wells of microplate in current detergent buffer to wash resin and remove unbound protein 3-5 times a. Add 150l buffer, and spin at 2000xg for 2min 5. Add 30l of new detergent to each well and spin at 2000xg for 2min 6. Repeat Step #5 five more times. On the sixth addition of buffer you can use the rest instead of 30l (approximately 40-50l). 7. Place the metal PCR Plate adapter on top of an elution plate and place this under the resin plate 8. Add 70l of elution buffer and spin at 2000xg for 2min 9. Assemble two more elution plates with the metal adapters, one with the Omscientia Low MWCO plate and the other with the Omscientia High MWCO plate. 10. Add 30l of the elution to one MWCO plate and 30l to the other MWCO plate and spin at 2000xg for 4 min Memb-PASS is patent-pending and available exclusively from Omscientia, Inc. 2010 Omscientia, Inc.

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a. If the elution volumes of the Omscientia Low MWCO plate seem low, spin again for another 2min Assemble the dot-blot apparatus with two pieces of pre-wet filter paper and pre-wet 0.2m nitrocellulose membrane Turn on the vacuum briefly to prime the apparatus and remove the water from the membrane Add 150l of 10% TCA (Tricholoacetic acid) to each well; do not turn on the vacuum before protein is added. Add 10l of the Omscientia Low MWCO elution to each well being sure to pipette the solution up/down several times while swirling the pipette tip in the well. This mixing/swirling is very important for nice blots, try to not scrape the nitrocellulose if at all possible. Turn on the vacuum until all the solution is removed from every well Add 150l PBS or 20mM Tris, 150mM NaCl buffer to each well Turn on vacuum until all the solution is removed from every well, you may have to pipette up and down to allow the solution to be fully removed. Repeat #16-17 two more times Disassemble the apparatus and briefly wash the membrane in ddH2O Block the membrane in 1X LI-COR blocking buffer for 30min (10 min if looking for speed). While blocking the 1st membrane, repeat the dot-blot (steps #11-20) for the Omscientia High MWCO elutions Add 15ml of Western Breeze Primary Antibody DIluent with Primary antibody added a. 21ml Water b. 6ml PartA c. 3ml PartB d. Mix above and add 4l IR-DYE antibody (1:8000 dilution) After 20min incubation (10 min if looking for speed), wash with Western Breeze Antibody Wash 4X (using 20ml each time) a. 150ml water b. 10ml Antibody wash solution Rinse the membrane with ddH2O several times Store membrane in 1X PBS until scanned on LI-COR or Fluorescence Imager Scan both membranes on the LI-COR or Fluorescence Imager with a scan intensity that does not saturate any of the spots Quantify the intensities of the spots using a median background correction Import the quantified spots into the Omscientia DFDA Analysis Master spreadsheet which can be downloaded from www.Omscientia.com To create the nice Quad Plot in Origin, import the data into the Omscientia Detergent rank plot Origin Project file which can be downloaded from www.Omscientia.com The Omscientia Differential Filtration Detergent Assay conditions can be downloaded from www.Omscientia.com

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Memb-PASS is patent-pending and available exclusively from Omscientia, Inc. 2010 Omscientia, Inc.