Radioimmunoprecipitation assay buffer
Radioimmunoprecipitation assay buffer is a lysis buffer used to lyse cells and tissue for the radio immunoprecipitation assay. This buffer is more denaturing than NP-40 or Triton X-100 because it contains the ionic detergents SDS and sodium deoxycholate as active constituents and is particularly useful for disruption of nuclear membranes in the preparation of nuclear extracts. The stronger detergents in RIPA buffer cause greater protein denaturation and decrease protein-protein interactions.
Recipe
RIPA buffer recipes vary slightly between authors and may include:- 10-50 mM Tris-HCl, pH 7–8
- 150 mM NaCl to keep the osmotic pressure near physiological
- nonionic detergents to prevent non-specific interactions between proteins or with the tube
- anionic detergents.
- Protease inhibitors or commercial protease inhibitor cocktail
- 1 mM each Na3VO4 and Na4P2O7 as phosphatase inhibitor
- 1 mM NaF as preservative
- 5-10 mM dithiothreitol or β-mercaptoethanol as a reducing agent.