RadioImmunoPrecipitation Assay (RIPA) Buffer, 10X

Preparation: Dilute this 10X buffer 10 times (e.g. 90 ml of deionized or distilled water + 10 ml of this buffer), mix well, and store 1 X buffer solution at 2-8oC for several weeks. Application: Lysis: Chill 1X buffer on ice add recommended PMSF, protease inhibitor cocktail and phosphatase inhibitor cocktail according to manufacture protocol. A. Adherent Cells: Wash cells with cold PBS to remove any traces of medium. Add 0.5 ml of 1X RIPA buffer/10 cm dish. Incubate plate on ice for 5 minutes. Scrape cells, transfer into mirocentrifuge tube, sonicate briefly. Centrifuge extracts for 10 minutes at 14,000 X g in a cold mirocentrifuge, save supernatant. B. Non-Adherent cells, wash cells pellet with cold PBS, centrifuge discard PBS, add approx. equal to cell pellet volume of RIPA buffer, sonicate briefly. Centrifuge extracts for 10 minutes at14,000 x g in cold mirocentrifuge, save supernatant.