Kta purifier 900 chromatography systems

Proteins were purified with Äkta purifier 900 chromatography systems (GE Healthcare, Helsinki, Finland). Cell-free extracts were loaded onto a Ni²⁺ Chelating HiTrapTM HP column (1–5 mL) (GE Healthcare, Finland) equilibrated with 50 mM potassium phosphate buffer, pH 7.2, at 1.0 mL/min. The column was washed with at least 3 volumes of the same buffer. Then the bound proteins were eluted with 0.5 M imidazole in 50 mM potassium phosphate buffer, pH 7.2. The fractions containing the purified enzyme were combined and dialyzed against the 50 mM potassium phosphate buffer, pH 7.2, at 4 °C overnight. Proteins were analyzed by SDS-PAGE according to Laemmli [66 (link)]. Protein concentration was determined by the Lowry method [67 (link)].

Proteins were purified with Äkta purifier 900 chromatography systems (GE Healthcare, Uppsala, Sweden). Cell-free extracts were applied to Ni²⁺ Chelating HiTrap HP column (GE Healthcare, Uppsala, Sweden) equilibrated with 50 mM potassium phosphate buffer, pH 7.0, at 1.0 mL/min. The column was washed with at least 3 volumes of the same buffer. Then the bound proteins were eluted with 0.5 M imidazole in 50 mM potassium phosphate buffer, pH 7.0. The fractions containing the purified enzyme applied to HiTrap Desalting column with Sephadex G-25 resin (GE Healthcare, Uppsala, Sweden) and eluted with 50 mM potassium phosphate buffer, pH 7.0. Proteins were analyzed by SDS-PAGE according Laemmli [35 (link)]. Protein concentration was determined by Lowry method [36 (link)].