PROTEIN EXTRACTION
1. For supernatants
Use the Nanosep® Centrifugal columns to concentrate the protein.
 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*.
* The desired concentrate volumes should be 25-35 µL.
 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
2. For cells grown in a 6 or 12-well plate:
 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*;
* 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).
** 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.
 Put plate on ice for 30 minutes and mix by shaking the plate every 10 minutes;
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 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).
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