Western Blot/Tissue Preparation

Overview
This is a protocol for preparing whole cell lysate from tissue, for western blot analysis.

RIPA Buffer
per 100ml: (final concentration)
 * 5ml of 1M Hepes, pH 7.6 (50 mM)
 * 200µL of 0.5M EDTA (1 mM)
 * 7 ml of 10% DOC (Na deoxycholate) (0.7%)
 * 10 ml of 10% NP-40 (IPGEL) (1%)
 * 10 ml of 5M LiCl or 2.12g powder (0.5 M)

Protease inhibitors

 * For example, Complete tabs from Roche
 * Or

Procedure

 * 1) Weigh tissue.
 * 2) Dice into very small pieces using a clean razor blade.
 * 3) Add 3 ml of ice cold RIPA buffer (supplemented with protease inhibitors) per gram of tissue.
 * 4) Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator. Maintain temperature at 4° C throughout all procedures.
 * 5) Incubate on ice for 30 minutes.
 * 6) Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4° C.
 * 7) Remove supernatant and centrifuge again. The super-natant fluid is the total cell lysate.

Contact

 * cmc