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. 2009 Jan 21;29(7):1972–1986. doi: 10.1128/MCB.01590-08

FIG. 3.

FIG. 3.

The affinity-purified anti-histone H3.3 antibody immunoprecipitates recombinant histone H3.3 but not recombinant histone H3. Recombinant H3 and H3.3 (25 μg/ml; New England BioLabs) were subjected to immunoprecipitation with 4 μg of either anti-histone H3 (ab1791; Abcam) or anti-histone H3.3 (generated in this investigation) antibody. As a negative control (−Ab), the immunoprecipitation was performed in the absence of either antibody. Alternatively, the anti-histone H3.3 antibody was preincubated with the H3.3-specific peptides that were synthesized by Auspep, Pty. Ltd., Australia. Each H3.3-specific peptide (CGGRKSAPSTGGVKK and CGGRKSAPS*TGGVKK) was made to a final concentration of 10 μM before incubation with the anti-histone H3.3 antibody for 0.5 h at room temperature with constant rotation. Subsequent immunoprecipitation with the anti-histone H3.3 antibody was performed as described above. Samples were then subjected to Western blotting with the anti-histone H3 antibody. Note that the anti-histone H3 antibody (ab1791; Abcam) raised against the C-terminal region of histone H3 is known to cross-react with both histones H3 and H3.3 because of the homology of this region in both proteins (Abcam, personal communication). The asterisk denotes the site of phosphorylation.