Kta pure 25 fplc system

Size-exclusion chromatography (SEC) was conducted using a Superose 6 10/300 GL column with ÄKTA pure 25 FPLC system (GE Healthcare Life Sciences, UK). Rh Bri3 BRICHOS samples were run at a flow rate of 0.5 ml·min⁻¹ using 20 mM PB, 0.2 mM EDTA at pH 8.0 or 20 mM ammonium acetate buffer, pH 8.0 and the eluates were monitored at 280 nm. Calibration curves from LMW and HMW Gel Filtration Calibration Kits (GE Healthcare, UK) were used for the determination of the hydrodynamic radius. Bri3 hydrodynamic diameter was derived from the Stokes radius obtained by comparison to a standard curve derived from elution volumes of standard proteins with known Stokes radii.
For analyses of CS and rh Bri3 BRICHOS complex formation, 500 μL of each sample were eluted in 20 mM ammonium acetate buffer, pH 8.0 using flow rate of 0.7 ml·min⁻¹.

The bacterial pellet was re-suspended in lysis buffer (50 mM Potassium phosphate, 10% glycerol, 30 mM Imidazole, 1% Tween, protease inhibitor, 1 μM FAD, 1 mM DTT, pH = 8) using 5 ml of buffer per gram of pellet. 30 ml cell suspension were sonicated for 5 min (5s on/10s off, 60% amplitude) with a sonicator (EpiShear, Active Motif) equipped with a ¼” microtip probe. The lysate was centrifuged (16000 x g, 4°C) for 30 min to pellet cell debris. An ÄKTA Pure 25 FPLC system, equipped with a 5 ml HisTrapFF Crude Column (GE Healthcare), was used for affinity purification. After column equilibration with 10 column volumes (CV) of wash buffer (50 mM Potassium phosphate, 10% Glycerol, 30 mM Imidazole, pH = 8), the supernatant was loaded onto the column and washed with 20 CV wash buffer. The protein was eluted with 2 CV elution buffer (50 mM Potassium phosphate, 10% Glycerol, 500 mM Imidazole, pH = 8). Imidazole was removed by dialysis and the protein was frozen in liquid nitrogen and stored at - 80°C until further use.