Size Exclusion Chromatography

The affinity chromatography purified protein is subjected to a size exclusion column (2.5 × 45 cm) packed with superdex75 (GE Healthcare) and equilibrated with lysis buffer. Protein concentration used for size exclusion chromatography is ~500 μM. Fractions of purified protein are eluted at a flow rate of 1 ml/min using a peristaltic pump (GE Healthcare). Absorbance at 280 nm wavelength is measured for all the fractions to identify the pure protein. Calibration of the column is performed using standard proteins namely, Lysozyme (14.4 KDa), Carbonic anhydrase (29 KDa), Ovalbumin (43 KDa), BSA (66 KDa) and Conalbumin (75 KDa). The error bars for standard proteins and STY3178 are estimated as standard errors from three sets of repeat experiments.