Resource s ion exchange column

The venom was a pooled sample obtained from three adult N. sumatrana captured in central Malaysia (Negeri Sembilan) and was supplied by Snake Valley (Seremban, Malaysia).
Resource S ion exchange column and HiTrap Protein A HP affinity column were purchased from GE Healthcare (New Jersey, USA). Goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate was obtained from Abnova, Taipei, Taiwan. Lichrosphere WP 300 C₁₈ reverse-phase column cartridge was purchased from Merck, New Jersey, USA. iBlot Gel Transfer stacks and iBlot blotting system were supplied by Invitrogen. Sephadex G-25 gel beads and all other reagents were purchased from Sigma – Aldrich (St. Louis, USA) or as stated in the methods.

BL21 gold cells were transformed with plasmid pAcKRS-3 encoding for the tRNA acetyl-lysine synthetase and pCDF PylT-1, and containing the amber suppressor tRNA and the H3 cDNA wild type, K64AMBER, or K9AMBER codon. The cells were then cultured as described before (Neumann et al., 2009 (link)). To purify the recombinant histones, cells were resuspended in wash buffer (50 mM Tris-HCl pH7.5, 100 mM NaCl, 1 mM β-mercaptoethanol) supplemented with 20 mM Nicotinamide (NAM) and 10 mg/ml lysozyme and then sonicated. The inclusion bodies were washed with the same buffer supplemented with 1% Triton X-100 and then histones were extracted with unfolding buffer (6 M Guanidium HCl, 20 mM Na-acetate pH5.2, 1 mM DTT) for 1 hr at RT. The extracted proteins were dialysed against SAU200 buffer (7 M urea, 20 mM Na-acetate pH5.2, 200 mM NaCl, 5 mM β-mercaptoethanol, 1 mM EDTA) before loading the sample on a ResourceS ion exchange column (GE Healthcare Life Sciences).