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Co-immunoprecipitation

BC Bi-He Cai
PW Po-Han Wu
CC Chi-Kan Chou
HH Hsiang-Chi Huang
CC Chia-Chun Chao
HC Hsiao-Yu Chung
HL Hsueh-Yi Lee
JC Jang-Yi Chen
RK Reiji Kannagi
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Cells were scraped into NP40 lysis buffer containing protease and phosphatase inhibitor cocktails (Merck). After cellular debris was removed by centrifugation, the pre-cleared extract was incubated with anti-FLAG M2 Magnetic Beads (Sigma) for 2–3 h at 4 °C using constant rotation. The beads were collected in magnetic separation rack and were washed three times in wash buffer (150 mM NaCl; 50 mM Tris, pH 7.5; 1 mM EDTA; 0.5% (w/v) NP40; 10% glycerol). Bound proteins were eluted with 20 μl of 500 μg/ml Flag peptide (Sigma) in elution buffer (150 mM NaCl; 50 mM Tris, pH 7.5; 1 mM EDTA; 0.05% NP40; 10% glycerol) for 10 min at 4 °C and were detected by using anti-p53 (sc-126, Santa Cruz) or anti-p73 (ab14430, Abcam) by western blot analysis.

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