Tnt coupled transcription translation kit

PBK and HPK fusion proteins (10 μg each) containing 6× His tags were prebound to nickel-nitrilotriacetic acid resin (10 μl 50% slurry) in 200 μl incubation buffer (50 mM NaH₂PO₄ [pH 8], 0.1 M NaCl, 5 mM imidazole, 10% glycerol, 0.01% NP-40) for 1 h at 4°C. Unbound protein was removed by washing the resin once in incubation buffer and twice in wash buffer (incubation buffer containing 0.05 M NaCl). [35S]Met-labeled PB produced by in vitro translation in accordance with the manufacturer's protocol (Promega TNT coupled transcription/translation kit) was added to each mix to achieve a 200-μl final volume in wash buffer and incubated with agitation for 2 h at 4°C. The resin was pelleted and washed four times in wash buffer, and the pelleted resin was resuspended in a mixture of 8 μl water and 8 μl SDS-PAGE loading dye. Mixtures were boiled for 5 min, followed by pelleting and loading of supernatants onto the gel.

GST-Dot1l was purchased from epicypher. GST and GST-Dot1l plasmids were transformed into BL21(DE3)E. coli(NEB) and production was induced by 0.1M IPTG treatment. Resultant proteins were purified using Glutathione Sepharose 4B (GE) according to the manufacturer’s recommended protocol. [³⁵S]-labeled Zc3h10 protein was produced by using TNT coupled transcription/translation kit (Promega). Of GST fusion proteins, 20 µg were incubated for 2 hr at 4°C with in vitro translated Zc3h10 and glutathione sepharose beads. The beads were washed three times with binding buffer, and bound proteins were eluted by boiling in Laemmli sample buffer, separated by SDS-PAGE and analyzed by autoradiography.