Cobalt column

Each 10⁸ High Five cell pellet was resuspended in 5 ml of lysis buffer A [50 mM Hepes (pH 7.4), 500 mM arginine, 1 mM EGTA/EDTA, and 1 protease inhibitor tablet mini (Roche)]. After centrifugation (150,000g for 30 min at 4°C), the supernatant was diluted 10 times in dilution buffer [50 mM Hepes (pH 7.4), 100 mM NaCl, 0.1% Triton X-100, and 1 mM EGTA/EDTA] and centrifuged again at 150,000g for 30 min at 4°C. The supernatant was passed through a 4-ml Q-Sepharose and loaded on a 5-ml SP Sepharose. After washing with Q/SP column buffer [50 mM Hepes (pH 7.4), 150 mM NaCl, and 0.05% Triton X-100], bound proteins were eluted with a five-column volume of SP elution buffer [50 mM Hepes (pH 7.4), 400 mM NaCl, and 0.05% Triton]. Imidazole (6 mM) was then added, and the solution was loaded on a cobalt column (1 ml; Thermo Fisher Scientific). The cobalt column was first washed with 50 mM Hepes (pH 7.4) and 400 mM NaCl in the presence of 20 mM imidazole, and the bound proteins were further eluted in the presence of 200 mM imidazole. The proteins were further purified using Superdex-200 columns as described above for His-MAP6.