Additional file 3.

Figure S3 - Characterisation of the novel H2AR29me2a antibody. (A) The indicated amounts of unmodified H2AR29 peptide, monomethylated H2AR29 peptide, asymmetrically dimethylated H2AR29 peptide (aa 25 to 34 of H2A) and asymmetrically dimethylated H2AR3 peptide (aa 1 to 8) were spotted onto a membrane and probed with the H2AR29me2a antibodies. This antibody specifically recognised the immunising peptide in peptide dot blots, but not the unmodified or the H2AR3 dimethylated peptide. (B) Full-length recombinant H2A and equal amounts of acid-extracted histones (see Coomassie staining below) from HeLa and Mcf7 cells were loaded onto an SDS-PAGE gel and blotted with the H2AR29me2-specific antibody. (Upper panel) In the western blot, the H2AR29me2 antibody specifically recognised endogenous histones. (C) SDS-extracted histones were loaded onto an SDS-PAGE gel. H2AR29me2 antibody was incubated with no competitor, with 1 pmol unmethylated peptide, or with H2AR29me2 peptide. The methylated, but not the unmethylated peptide, competed for the signal, confirming the specificity of the antibody. In addition, the H3R17me2 peptide was not able to compete, showing that this antibody is specific for H2AR29me2. (D) Total nuclear extract (NIH-3T3 cells) was loaded onto an SDS-PAGE gel and (left panel) probed with H2AR29me2-specific antibodies; (right panel) Ponceau stain. In this whole-cell extract, the H2AR29me2 antibody specifically recognised H2A.

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Waldmann et al. Epigenetics & Chromatin 2011 4:11   doi:10.1186/1756-8935-4-11