Remove
media.
Wash
cells in PBS.
Add
1 ml RIPA to 100 mm dish.
Scrape
cells; draw cells up in 1 ml syringe with 21 or 22 gauge needle (protocol calls
for 21, I’ve been using 22), and dispense into microfuge tube. Draw up and down through needle 3 or 4 times
to shear DNA.
Let
sit on ice for 30-60 minutes.
Spin
at 15000 x g for 20 minutes at 4 C.
Save supernatant as whole cell lysate for Westerns.
RIPA buffer:
1X PBS
1% TritonX100
0.5% sodium deoxycholate
0.1% SDS
1 Complete mini protease
inhibitor tablet/10 ml, added right before use to cold RIPA.