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Co-immunoprecipitation and immunoblotting

YL Yool Lee
YS Yang Shen
LF Lauren J. Francey
CR Chidambaram Ramanathan
AS Amita Sehgal
AL Andrew C. Liu
JH John B. Hogenesch
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At 48 post-transfection of U2OS cells, cell lysates were harvested in radioimmunoprecipitation assay (RIPA) buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail (Roche). Protein-G coated magnetic beads (10004D, Life Technologies) were pre-incubated with 3 µg of anti-GFP antibody (G1544, Sigma) at 4 °C for 6 h. Antibody conjugated beads were incubated with equal amounts of total protein at 4 °C overnight. The final immune complexes were analyzed by immunoblot assay.

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