Determination of αH3K56me3 specificity and suitability in diverse applications.

<p>(A) Immunoblot peptide competition experiment. αH3K56me3 antibody was preincubated with competitor peptides before addition to immunoblots containing recombinant H3 protein (R) or acid extracted HeLa Kyoto histones (H) (top). Ponceau staining (bottom) serves as loading control. (B) IF microscopy peptide competition experiment. αH3K56me3 antibody (green) was preincubated with competitor peptides before addition to fixed HeLa Kyoto cells. DAPI (blue) stains DNA. Scale bar = 5 µm. (C) Spot-blot with different concentrations (5–1000 ng) of H3 peptides to determine αH3K56me3-binding affinities. (D) Immunoblot of sequential tryptic digest of HeLa Kyoto-derived mononucleosomes using αH3K56me3 (top), αH3K9me3 (middle) and αH3 (bottom). FL = full-length histone H3, GD = N-terminally deleted globular domain of histone H3.