Human histones H2A(K119C), H2B, H3.2(C110A), H3.2(C110A,K36C) and H4 were expressed and purified as previously reported (34 (link)). Histone heterodimer, H2A(K119C) and H2B, were then refolded separately from tetramer, H3.2(C110A) and H4 or H3.2KC36me3(C110A) and H4, by dialysis from unfolding buffer (7 M guanidine–HCl, 10 mM Tris–HCl pH 7.5, 10 mM DTT) to refolding buffer (5 mM PIPES pH 6.1, 2 M NaCl).
Heterodimer was then labeled with Cy5-maleimide (GE Healthcare) and purified as previously reported (11 (link),36 (link)–38 (link)). Heterodimer and tetramer were then combined to a molar ratio of 1:2.2 tetramer:heterodimer. The proteins were allowed to complex overnight at 4°C while gently rotating. The resulting octamer was then purified by size exclusion chromatography with a superdex 200 column. Histone octamer was concentrated in a 30 kDa MWCO Amicon Ultra (Millipore) and stored on ice.
Heterodimer was then labeled with Cy5-maleimide (GE Healthcare) and purified as previously reported (11 (link),36 (link)–38 (link)). Heterodimer and tetramer were then combined to a molar ratio of 1:2.2 tetramer:heterodimer. The proteins were allowed to complex overnight at 4°C while gently rotating. The resulting octamer was then purified by size exclusion chromatography with a superdex 200 column. Histone octamer was concentrated in a 30 kDa MWCO Amicon Ultra (Millipore) and stored on ice.
