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.

RIPA buffer is a lysis buffer used to lyse cells and tissues for RadioImmunoPrecipitation assay (RIPA).This buffer in addition to Triton X100 contains ionic detergents SDS and sodium deoxycholate which dissolves nuclear membranes and it also contains EDTA and EGTA. It is a low background producing buffer that can denature kinases. This buffer is not suitable to disrupt protein: protein interaction. Protease inhibitors e.g. PMSF, protease cocktail and phosphatase inhibitors cocktail should be added fresh to RIPA buffer.