Protein Purification via Gel Filtration

A Superose 6 Increase 10/300 GL gel filtration column (24 ml bed, GE Healthcare Life Sciences, Pittsburgh, PA) was equilibrated with F-buffer (0.1 M KCl, 2 mM MgCl₂, 0.2 mM dithiothreitol, 20 mM HEPES-KOH, pH 7.5). Proteins at 1.0 mg/ml (500 μl) were filtrated through a Spin-X 0.22 mm filter (Corning Costar, Tewksbury, MA), applied to the column, and eluted with the same buffer. The eluates were collected in fractions of 0.5 ml each and analyzed by SDS-PAGE. The gels were stained with Coomassie Brilliant Blue R-250 (National Diagnostics), scanned by an Epson Perfection V700 Photo Scanner at 300 dots per inch, and analyzed by densitometry using ImageJ. Correlation of elution profiles and dimeric/monomeric states of MBP-CAS-2C and MBP-CAS-2CΔβ8β9 was determined previously by dynamic light scattering (Iwase and Ono 2016 (link)).