Superdex 75 10 300 gl size exclusion column

The pET21b plasmid expressing the A3B-CTD (residues 187–382) construct was a courtesy of the Gronenborn Lab from University of Pittsburg School of Medicine. The A3B-CTD expression and purification procedure was adapted from published protocols with modifications (Byeon et al., 2016 (link)). Briefly, the plasmid was transformed into BL21-Gold (DE3) Competent Cells (Agilent). The bacteria cells were grown in LB broth containing 100 mg/L ampicillin to an OD600 of 0.8 and was then transferred to 16°C followed by protein expression induction overnight with 0.4 mM IPTG. The cells were pelleted and resuspended in Buffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 30 mM imidazole) and lysed using a cell disruptor. The supernatant was clarified by centrifugation at 16,000 RPM for 45 min and was applied to Ni-sepharose 6 fast flow resin (Cytiva). The resin was washed with 20 column volumes of Buffer A. The protein was eluted with Buffer B (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 300 mM imidazole). The eluate was concentrated and further purified with a Superdex 75 10/300 GL size exclusion column (Cytiva) pre-equilibrated with buffer containing 20 mM Tris-HCl pH 7.5, 100 mM NaCl. Peak fractions containing A3B-CTD were pooled and concentrated to ∼4 mg/mL for the assay.