Affinity Purification of Histone Binders

An amino propyl linker was added for coupling UNC0638^{26 (link)} to ECH sepharose 4B beads (General Electric cat# 17-0571-01). UNC0638 immobilized on sepharose beads were washed in Buffer D with 0.1% Triton-X100 twice before added into the nuclear extract. After incubation overnight, the beads was washed with 250mM KCl buffer (20mM HEPES pH7.9, 0.2mM EDTA, 250mM KCl, 20% Glycerol, 1mM PMSF, 1mM DTT and protease inhibitor cocktail) for five times. The pull-down products were eluted by boiling the beads in 2×SDS-PAGE loading buffer for 10 minutes. For histone peptide pull-down experiments, histone H3 N-tail peptides was synthesized with a biotin tag, then the peptide was conjugated to streptavidin beads and the pull-down experiment was carried out in a similar way as UNC0638 pull-down except for that the washing buffer was substituted by a low-stringent buffer containing 100mM KCl. The pull-down products were resolved by SDS-PAGE, either for immunoblotting or followed by in-gel tryptic digestion prior to MS analysis.