Western Blot/Tissue Preparation
< Western Blot(Difference between revisions)
This is a protocol for preparing whole cell lysate from tissue, for western blot analysis.
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)
- Weigh tissue.
- Dice into very small pieces using a clean razor blade.
- Add 3 ml of ice cold RIPA buffer (supplemented with protease inhibitors) per gram of tissue.
- Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator. Maintain temperature at 4° C throughout all procedures.
- Incubate on ice for 30 minutes.
- Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4° C.
- Remove supernatant and centrifuge again. The super-natant fluid is the total cell lysate.
- If necessary, use a longer centrifugation to obtain a clear lysate.