以下这是protocol Immunoprecipitation by M2 antibody M2 bind to FLAG tag is okay with high salt but no good with NP40 1. Wash transfected cells with cold PBS twice and lyse in lysis buffer (25mM Tris-HCl, pH 7.0, 300mM NaCl, 1% Triton X-100, containing 1mM PMSF, 1x Protease inhibitor cocktail [Roche]: 10贡g/ml each of aprotinin , leupeptin, and pepstatin) put on ice for 30min. 2. Clean cell lysate by centrifuge at 12000rpm for 20 min at 4度 and supernatants are used as cell extracts. Measure the protein concentration. One reaction 500贡g lysate. 3. Preclean the cell extracts by incubating with mouse IgG (if you do not have it, I used anti-mouse 2nd Ab instead) and protein-G sepharose (20microliter) at 4度 for 1 hour, then get rid of protein-G by centrifuge at 5000rpm 3 min at 4度. 4. Incubate 500贡g precleaned lysate with 2贡g M2 in eppendorf tube at 4度 overnight. 5. Add 20贡l protein-G sepharose to Rex4 incubate at 4oC for 1 hour. 6. The immunoprecipitates are washed 4-5 times with 1ml TBS-T buffer (centrifuge at 5000rpm 3 min at 4度, keep the beads by carefully discarding the supernatant). 7. Adding the SDS loading dye and heating at 95度 for 3 min then proceed the western blotting. TBS-T buffer: 50mM Tris-HCl, pH 7.5 1mM EDTA 150mM NaCl 0.05% Tween 20 全部的布骤只差在proteinＧ改用protein A~~家里只有protein A然後老板说没差 然後我要看的是protein protein interaction ip是用flag western是用A protein的单株抗体~老鼠的 impute OK都有东西 且ip若是A protein的单株抗体而western也是A protein的单株抗体~~确定有东西 但是ip flag 而western是 A protein的单株抗体则无任何interaction ~~不过确定会interaction~~别人的paper已经发表了~~所以我没做出来 我是没做ip flag且 western flag 但我不知protein G换成proteinA是否会影响FLAG的binding 请做过的人给我指教 --

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