以下這是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 全部的布驟只差在proteinＧ改用protein A~~家裡只有protein A然後老闆說沒差 然後我要看的是protein protein interaction ip是用flag western是用A protein的單株抗體~老鼠的 impute OK都有東西 且ip若是A protein的單株抗體而western也是A protein的單株抗體~~確定有東西 但是ip flag 而western是 A protein的單株抗體則無任何interaction ~~不過確定會interaction~~別人的paper已經發表了~~所以我沒做出來 我是沒做ip flag且 western flag 但我不知protein G換成proteinA是否會影響FLAG的binding 請做過的人給我指教 --

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