<p> PROTEIN EXTRACTION</p><p>1. For supernatants</p><p>Use the Nanosep® Centrifugal columns to concentrate the protein.</p><p> Rinse the membranes of the columns with PBS 1X by centrifugation during 6 minutes at 14000g at 4oC;  Discard the PBS and repeat the rinse; DO NOT LET THE MEMBRANES DRY OUT!!  Pipette 500 µL of the supernatants into the columns and centrifuge during 12 minutes at 14000g at 4oC;  Check the volumes of supernatants left on the membranes by carefully pipetting (in an angle) the concentrate volumes. If necessary, centrifuge again (and many times more if necessary) for 2-3 minutes until achieving the desired concentrate volumes*.</p><p>* The desired concentrate volumes should be 25-35 µL.</p><p> Transfer the samples into new eppendorf tubes and add PBS 1X if needed to bring the volumes of all samples to the same final volume;  Add 5X loading dye to the samples for a 1:5 dilution factor in the total volume (example: for 31 µL of sample, add 8 µL of loading dye + 1 µL of PBS 1X → total volume = 40 µL in which 8/40 = 1:5 dilution factor).  Vortex the samples and centrifuge for a few seconds.  Boil the samples for 5 minutes.  Load the samples or store them at -80oC</p><p>2. For cells grown in a 6 or 12-well plate:</p><p> Remove the media and rinse the cells in the well with 2 or 1 mL of cold PBS 1X ;  Add 200 or 100 µL of ice cold lysis buffer containing protease inhibitors 1X*;</p><p>* Prepare previously by adding 100 µL of protease inhibitors 7X** (aliquots in box at -20oC on Severine shelf, lab 216) to 600 µL of cold lysis buffer (small bottle at 4oC in drawer of WB, lab 216).</p><p>** To prepare aliquots of protease inhibitors 7X, dissolve a tablet of protease inhibitors (small metallic bottle at 4oC in drawer of WB, lab 216) into 1.5 mL of cold lysis buffer; once dissolved, aliquot into 100 µL volume.</p><p> Put plate on ice for 30 minutes and mix by shaking the plate every 10 minutes;</p><p>1  Homogenize the lysate well, transfer to an eppendorf tube;  Centrifuge the samples 15 minutes at 12000g (12300 RPM) at 4oC;  Transfer the supernatant into a new eppendorf tube taking care not to disturb the pellet;  Store the lysate at -20oC (several months).</p><p>2</p>