Western Blot/Tissue Preparation

Overview

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

Materials

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 [1]
• Or [2]

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.

Notes

• If necessary, use a longer centrifugation to obtain a clear lysate.

Contact

• cmc