Immunoprecipitation Conditions

Wash Buffer

50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% NP-40, pH 7.4.

For immunoprecipitation of phosphorylation-dependent epitopes, phosphatase inhibitors may be added to the wash buffer. We recommend adding one or more of: 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 10 mM NaF, or 1-2 mM EGTA.

Whole Cell Lysis Buffer

50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% NP-40,  1 mM sodium orthovanadate, 0.25 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL chymostatin, pH 7.4

Nuclear Extract Lysis Buffer

10 mM HEPES, 1.50 mM MgCl₂, 10 mM KCl, 0.1% NP-40,  1 mM sodium orthovanadate, 0.25 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL chymostatin, pH 7.9

SDS Sample Buffer

0.25 M Tris, 6% SDS, 10% glycerol, 20 mM dithiothreitol, bromophenol blue, pH 6.8