Baf155 antibody

For T7- and Flag-IP experiments, HEK293T cells were transiently transfected for 24 hr with indicated plasmids, along with GFP to tell transfection efficiency, using calcium phosphate transfection. Cells were lysed in Lysis Buffer, sonicated, and debris cleared by centrifugation. Equal amounts of lysates were used for immunoprecipitation with T7 antibody or, in the case of Flag-IP, M2-conjugated agarose beads (Sigma) overnight at 4°C. For T7-IPs, antibody complexes were bound to protein G agarose (Sigma) the next day. Following binding, all IP samples were washed extensively in Lysis Buffer and then boiled in loading dye. For endogenous BAF155 co-immunoprecipitation experiments, samples were processed similarly, except cells were treated for 1.5 hr with 25 μM MG132 (VWR) and then nuclei from cells were extracted in 10mM HEPES, pH 7.9, 10mM KCl, 0.4% NP-40 prior to lysis in Lysis Buffer. Equal amounts of nuclear lysates were subjected to immunoprecipitation with 5 μl of BAF155 antibody (Cell Signaling, 11956) overnight at 4°C and bound to protein A agarose (Roche) the next day. Following four washes as described above, samples were boiled in loading dye.