Radioimmunoprecipitation assay buffer (RIPA buffer) is a

lysis buffer A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells (e.g. western blot for protein, or for DNA extraction). Most lysis buffer ...

used to

lyse Lyse may refer to: People * Lyse Doucet (born 1939), Canadian journalist, presenter and correspondent for BBC World Service radio and BBC World television * Lyse Richer (born 1958), Canadian administrator and music teacher * Carl L. Lyse (1899– ...

cells and tissue for the

radio Radio is the technology of communicating using radio waves. Radio waves are electromagnetic waves of frequency between 3 hertz (Hz) and 300 gigahertz (GHz). They are generated by an electronic device called a transmitter connec ...

immunoprecipitation Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sam ...

assay An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity ...

(RIPA). This buffer is more denaturing than

NP-40 NP-40 (also known as Tergitol-type NP-40 and nonyl phenoxypolyethoxylethanol) is a commercially available detergent with CAS Registry Number 9016-45-9. NP-40 is an ethoxylated nonylphenol for non-ionic surfactants and can act as emulsifier and de ...

Triton X-100 Triton X-100 (''n'') is a nonionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic group. The hydrocarbon group is a 4-( 1,1,3,3-tetr ...

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 (such as SDS) cause greater protein denaturation and decrease protein-protein interactions.

Recipe

RIPA buffer recipes vary slightly between authors and may include: * 10-50 mM

Tris Tris, or tris(hydroxymethyl)aminomethane, or known during medical use as tromethamine or THAM, is an organic compound with the formula (HOCH2)3CNH2. It is extensively used in biochemistry and molecular biology as a component of buffer solutions ...

-HCl (10 mM

sodium phosphate A sodium phosphate is a generic variety of salts of sodium () and phosphate (). Phosphate also forms families or condensed anions including di-, tri-, tetra-, and polyphosphates. Most of these salts are known in both anhydrous (water-free) and ...

may be used instead), pH 7–8 * 150 mM NaCl to keep the osmotic pressure near physiological * nonionic detergents (1% Triton X-100 or

) to prevent non-specific interactions between proteins or with the tube * anionic detergents (0.1-0.5% deoxycholate, 0.1-0.5% SDS). The following ingredients are optional and included as needed: *

Protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

inhibitors (1 mM

PMSF In biochemistry, phenylmethylsulfonyl fluoride (PMSF) is a serine protease inhibitor (serine hydrolase inactivator) commonly used in the preparation of cell lysates. PMSF does not inactivate all serine proteases. The effective concentration of P ...

(fresh from 1 M stock in i-propanol), 1 μg/mL each

leupeptin Leupeptin, also known as ''N''-acetyl-L-leucyl-L-leucyl-L-argininal, is a naturally occurring protease inhibitor that can inhibit cysteine, serine and threonine peptidases. It is often used during ''in vitro'' experiments when a specific enzymati ...

aprotinin The drug aprotinin (Trasylol, previously Bayer and now Nordic Group pharmaceuticals), is a small protein bovine pancreatic trypsin inhibitor (BPTI), or basic trypsin inhibitor of bovine pancreas, which is an antifibrinolytic molecule that inhibi ...

pepstatin Pepstatin is a potent inhibitor of aspartyl proteases. It is a hexa-peptide containing the unusual amino acid statine (Sta, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid), having the sequence Isovaleryl-Val-Val-Sta-Ala-Sta (Iva-Val-Val-Sta-Al ...

, 1-5 mM

EDTA Ethylenediaminetetraacetic acid (EDTA), also called EDTA acid, is an aminopolycarboxylic acid with the formula . This white, slightly water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-solubl ...

, 0.5-1 mM EGTA, 5 mM

aminocaproic acid Aminocaproic acid (also known as ε-aminocaproic acid, ε-Ahx, or 6-aminohexanoic acid) is a derivative and analogue of the amino acid lysine, which makes it an effective inhibitor for enzymes that bind that particular residue. Such enzymes inc ...

) 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 * 5-10 mM

dithiothreitol Dithiothreitol (DTT) is an organosulfur compound with the formula . A colorless compound, it is classified as a dithiol and a diol. DTT is redox reagent also known as Cleland's reagent, after W. Wallace Cleland. The reagent is commonly used in ...

(DTT) or

β-mercaptoethanol 2-Mercaptoethanol (also β-mercaptoethanol, BME, 2BME, 2-ME or β-met) is the chemical compound with the formula HOCH2CH2SH. ME or βME, as it is commonly abbreviated, is used to reduce disulfide bonds and can act as a biological antioxidant by sc ...

as a reducing agent.

References

Laboratory techniques {{biology-stub