Radioimmunoprecipitation assay buffer

Radioimmunoprecipitation assay buffer (RIPA buffer) is a lysis buffer used to lyse cells and tissue for the radio immunoprecipitation assay (RIPA).[1][2] 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 RIPA buffer gives low background but can denature kinases.

Recipe

RIPA buffer recipes vary slightly between authors and may include:

  • 10-50 mM Tris-HCl (10 mM sodium phosphate may be used instead), pH 7–8
  • 150 mM NaCl to keep the osmotic pressure near physiological
  • nonionic detergents (1% Triton X-100 or NP-40) to prevent non-specific interactions between proteins or with the tube
  • anionic detergents (0.1-0.5% deoxycholate, 0.1-0.5% SDS). This needs to be optimised for every assay, the higher the concentration, the cleaner the result, but the lower the signal.

The following ingredients are optional and included as needed:

  • Protease inhibitors (1 mM PMSF (fresh from 1 M stock in i-propanol), 1 µg/ml each leupeptin, aprotinin, pepstatin, 1-5 mM EDTA, 0.5-1 mM EGTA, 5 mM aminocaproic acid) or commercial protease inhibitor cocktail (use according to the manufacturer's instruction)
  • 1 mM each Na3VO4 and Na4P2O7 as phosphatase inhibitor (if phosphorylation status is important)
  • 1 mM NaF as preservative (very toxic!)
  • 5-10 mM dithiothreitol (DTT) or β-mercaptoethanol as antioxidant. DTT is more expensive than βME, but its redox potential is better suited for the Cys-Cys bond.

References
  1. Alcaraz C, De Diego M, Pastor MJ, Escribano JM (July 1990). "Comparison of a radioimmunoprecipitation assay to immunoblotting and ELISA for detection of antibody to African swine fever virus". J. Vet. Diagn. Invest. 2 (3): 191–6. doi:10.1177/104063879000200307. PMID 2094444.
  2. Ngoka LC (October 2008). "Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers". Proteome Sci. 6 (1): 30. doi:10.1186/1477-5956-6-30. PMC 2600628. PMID 18950484.


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