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[]

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