Gel filtration experiments were performed using Superdex75 HR 10/30 size exclusion column (GE Healthcare) with a separation range of 3–70 kDa connected to an FPLC prime automated liquid chromatography system (Amersham Biosciences). The running buffer used was 10 mM Tris-HCl pH 8, 200 mM NaCl. The column was calibrated using gel filtration molecular weight standard (Bio-Rad). The following standards were used for calibration: Thyroglobulin (670 kDa, Rs = 8.6 nm), γ-globulin (158 kDa, Rs = 5.1 nm), Ovalbumin (44 kDa, Rs = 2.8 nm), Myoglobin (17 kDa, Rs = 1.9 nm), Vitamin B12 (1.35 kDa), and Dextran blue (2 MDa).
A 0.5 ml protein sample at a final concentration of 5 µM was filtered and chromatographically analyzed using a flow rate of 0.5 ml/min. Absorbance was monitored at 280 nm, elution volumes were determined from UV chromatogram. The partition coefficient, Kav, was calculated from the elution volume of the sample, Ve, and total bed volume, Vt, using the expression: . Stokes radius (Rs) for all proteins was calculated by plotting versus known Rs. Calibration curves and equations were established.
A 0.5 ml protein sample at a final concentration of 5 µM was filtered and chromatographically analyzed using a flow rate of 0.5 ml/min. Absorbance was monitored at 280 nm, elution volumes were determined from UV chromatogram. The partition coefficient, Kav, was calculated from the elution volume of the sample, Ve, and total bed volume, Vt, using the expression: . Stokes radius (Rs) for all proteins was calculated by plotting versus known Rs. Calibration curves and equations were established.