Hiload superdex 200 16 600 pg columns

Bacterial pellets expressing the Ame1-Okp1 complex (AO) were resuspended in lysis buffer (30 mM HEPES, pH 7.5, 30 mM imidazole [A] or 1 M imidazole [B], 600 mM NaCl, and 5 mM β-mercaptoethanol). AO loaded on the HisTrap HP 5 ml column was washed with 30 CV lysis buffer before eluted with 300 mM imidazole in the otherwise same buffer or using an imidazole gradient from 0% to 60%. AO eluted between 150 mM and 300 mM imidazole. Subsequently, the complex was purified over gel filtration Superdex 200 10/300 GL or HiLoad Superdex 200 16/600 pg columns (GE Healthcare) in 30 mM HEPES, pH 7.5, 250 mM NaCl, 5% glycerol, and 2 mM TCEP. The complex eluted in two peaks from the column, of which the later eluting peak was used for subsequent experiments. All used amino acid substitutions were purified in the same way as described above.

Bacterial pellets expressing the AO complex (AO) were resuspended in lysis buffer (30 mM HEPES, pH 7.5, 30 mM imidazole, 600 mM NaCl, and 5 mM β‐mercaptoethanol). AO loaded on the HisTrap HP 5‐ml column was washed with 30 CV lysis buffer before eluted with 300 mM imidazole in the otherwise same buffer. Subsequently, the complex was purified over gel filtration Superdex 200 10/300 GL or HiLoad Superdex 200 16/600 pg columns (GE Healthcare) in 30 mM HEPES, pH 7.5, 250 mM NaCl, 5% glycerol, and 2 mM TCEP. The complex eluted in two peaks from the column, of which the later eluting peak was used for subsequent experiments.