- Rinse a 60 mm culture dish of confluent cells with PBS.
- Lyse the cells with 0.5 ml cold Immunoprecipitation Buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM sodium ortho-vanadate, 0.2 mM PMSF, 1% Triton X-100, 0.5% NP-40).
- Maintain constant agitation for 30 minutes at 4°C.
- Scrape the cells from the dish. Sonicate on ice for 5 seconds; repeat 4 times. Centrifuge for 5 minutes at 4°C. The supernatant is the crude cell lysate. Assay for total protein then adjust concentration to approximately 1 mg/ml with Immunoprecipitation Buffer.