Stripablot stripping buffer

For co-immunoprecipitation experiments, HeLa cells were grown in 10 cm Petri dishes and transfected with the indicated constructs as described above. After 36 hours, cells were lysate in an appropriate volume of lysis buffer (125 mM NaCl, 25 mM TRIS-Cl pH7.4, 1 mM EGTA-Tris pH 7.4, 0.5% n-Dodecyl β-D-maltoside, Complete EDTA free protease inhibitor mixture and PhoSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science)). Whole cell lysate was precleared for 30’ with a control agarose resin (Pierce). 1 mg of pre-cleared proteins from the indicated conditions was incubated with monoclonal αFlag or αHA agarose-conjugated antibody (Sigma-Aldrich) and the co-immunoprecipitation was performed following manufacturer instructions. After three 10’ washes in lysis buffer, the bait was eluted in a non-reducing Laemli sample buffer and denatured for 10’ at 90°C. The precleared lysate (Input) and the eluted fraction (CoIP) were separated by SDS-PAGE, transferred to Hybond-C Extra membrane (Amersham) and developed according to standard procedures. The same membrane in each panel was stripped for 10’ in StripABlot stripping buffer (Euroclone) and probed with different antibodies as indicated.