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.
Recombinant Histone Octamer Assembly
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.
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Corresponding Organization : The Ohio State University
Other organizations : University of Colorado Denver
Protocol cited in 1 other protocol
Variable analysis
- Histone variant used for heterodimer and tetramer formation (H2A(K119C), H2B, H3.2(C110A), H3.2(C110A,K36C), H4)
- Octamer formation and purification by size exclusion chromatography
- Refolding buffer (5 mM PIPES pH 6.1, 2 M NaCl)
- Unfolding buffer (7 M guanidine–HCl, 10 mM Tris–HCl pH 7.5, 10 mM DTT)
- Molar ratio of 1:2.2 tetramer:heterodimer for octamer formation
- Incubation temperature of 4°C for overnight complexation
- None specified
- None specified
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