Whole Cell Lysates from Monolayer Cultures FGB 8/02

From tissue culture dishes

Remove media.

Wash cells in PBS.

Add 1 ml RIPA (10-cm dish), 100 ul (6-well dish), or 50 ul (12-well dish).

Scrape cells then add to a 1.5 ml tube

Draw cells up in 1 ml syringe with 21 or 22-gauge needle

Draw cell lysis 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.

Quantitate using Albumin standards and BCA kit.

From Cell pellet

After pellet cells, aspirate supernatant, wash in PBS if desired, then add RIPA buffer and continue as above.

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.

RIPA is as follows:

10mL PBS, 100ul TritonX100, 0.05g sodium deoxycholate (add first on scale into 15mL tube), 10ul of 10%SDS, and 1 B.M. Complete Mini protease inhibitor tablet (Roche #1836153).