1. Purify Flag-H2A from HeLa cells stably expressing Flag-H2A and purify Flag-H2B from T85 yeast strain (provided by Dr. Shelley L. Berger) and YKH045 yeast strain (provided by Dr. Mary Ann Osley) with anti-Flag IP under denature conditions(Wang et al., 2006; Robzyk, K, 2000).
2. Mix histone H2B purified from these two yeast strains at appropriate ratio to match the ratio of uH2A/H2A that were purified from HeLa cells, as judged by Coomassie blue staining. 3. Purify recombinant H3, H4, H2A, and H2B from E. coli as described (Luger et al., 1999).
4. PCR amplify rhe 5s DNA fragments from the XP-10 plasmid (provided by Dr. Jeffrey J. Hayes) and the 5’ primer used for PCR amplification was biotinylated (Wang et al., 2006).
5. Perform histone octamer refolding and nucleosome reconstitution as described (Dyer et al., 2004).
6. To purify individual H2A, uH2A, H2B, uH2B, resolve the eluate from anti-Flag IP on a SDS-PAGE and excise gel slices containing H2A, uH2A, H2B and uH2B.
7. After extensive washing, electro-elute proteins out from these gel slices and further passed through a hydroxyapatite column to remove the excess SDS.
8. Combine fractions from the hydroxyapatite column and dialyze against water at 4 °C overnight and then lyophilize.
9. Use an aliquot to determine the amount of proteins as revealed by Coomassie blue staining. After dissolving in histone unfolding buffer (6 M guanidinium chloride, 20 mM Tris-Hcl, pH 7.5, 5 mM DTT), perform histone octamer refolding and nucleosome reconstitution as described (Dyer et al., 2004).