Protein Size-Exclusion Chromatography

Samples were injected 70 microliters per run onto a Superdex 200 10/300 GL (24 ml bed volume; GE Healthcare) and run at 4°C and 0.38 ml/min in standard column buffer (100 mM NaCl, 20 mM Tris–HCl, (pH 8), 1 mM DTT, 0.1 mM EDTA, 5% glycerol). Elution volume was averaged from multiple independent runs with purified EcoWI protein (2.7 mg/ml). The partition coefficient (K_av) of EcoWI was determined by K_av = (v_e – v_o)/(v_t – v_o) where v_e is the elution volume, v_o is the void volume of the column and v_t is the total column volume. v_o and v_t were determined empirically by the addition of blue dextran and DTT, respectively, to the sample. A standard curve was created by obtaining the K_av value of the standard proteins carbonic anhydrase (29 kDa), ovalbumin (43 kDa), conalbumin (75 kDa), and Blue Dextran (GE Healthcare) run under the same condition, as described previously (Xu et al., 2011).