Histrap hp ni2 column

Recombinant protein (200 μM) was incubated with 5 mM dithiothreitol (DTT) for 1 h at room temperature. DTT was removed via two consecutive passes through a PD-10 Sephadex G-25 M buffer exchange column (GE Healthcare) according to the manufacturer’s instructions into labeling buffer (50 mM HEPES [pH 7.1], 200 mM KCl, 5% glycerol, 120 μM TCEP). Flowthrough was analyzed for protein content at 280 nm. Reduced protein (50 μM) was incubated with 100 μM ATTO 488-maleimide (ATTO-TEC) overnight at 4°C, shielded from light, and subjected to gentle shaking. The reaction was stopped by the addition of 6 mM β-mercaptoethanol, and mixtures were applied to a 1-ml HisTrap HP Ni²⁺ column (GE Healthcare) before elution using a gradient of buffer B (50 mM Tris [pH 7.5], 200 mM NaCl, 5% glycerol, 500 mM imidazole) and subsequent dialysis to remove the imidazole. Labeling efficiency was calculated in accordance with the fluorescent dye manufacturer’s guidelines.

Proteins were purified from 1–2 L E. coli cultures. Cultures were grown to an OD₆₀₀ 0.5–0.7, expression induced with 1 mM IPTG and incubated for 3 h at 37°C. Protein purifications were performed by either nickel or glutathione affinity chromatography. For nickel purifications, cell pellets were resuspended in 5 ml Buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM imidazole), lysed with 100 μg lysozyme and sonication and the filtered cell lysate loaded onto a 1 ml HisTrap HP Ni²⁺ column (GE Healthcare) before elution using a gradient of Buffer B (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 500 mM imidazole). GST-tagged proteins were resuspended in 5 ml PBS, lysed and the cell lysate was loaded onto a 1 ml GSTrap HP column (GE Healthcare) before elution using 50 mM Tris pH 8.0, 10 mM reduced glutathione. Protein containing fractions were dialysed in 50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol before storage at -80°C. Protein concentrations were determined by A₂₈₀ readings.

Proteins were purified from 500 mL E. coli BL21 DE3 cultures. Cultures were grown at 37°C to an OD₆₀₀ of 0.5 to 0.7; expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated for 3 h at 30°C. Cell pellets were resuspended in 5 mL Buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM imidazole) and lysed by sonication upon addition of 20 μg/mL lysozyme. Protein purifications were performed by nickel affinity chromatography. The filtered cell lysate was loaded onto a 1-mL HisTrap HP Ni²⁺ column (GE Healthcare) before elution using a gradient of Buffer B (50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 500 mM imidazole). Protein-containing fractions were dialyzed in 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5% glycerol before concentrating using a 50-kDa centrifugal filter (Thermo Scientific) and storage at −80°C. For analysis of dimer formation, samples were mixed with a loading dye lacking β-mercaptoethanol and were not heated prior to analysis on SDS-PAGE. For use in Gmk enzymatic assays, proteins were further purified by size exclusion chromatography using a preparative 16/60 Superdex 200 column and a 50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol buffer system.