Gel filtration chromatography of serum proteins

All chemicals were obtained from SIGMA (St. Louis, MO, USA). To obtain fractions with various molecular weights, serum samples were subjected to gel filtration chromatography as described [19] with minor modifications: A 7 ml column of Sephacryl S-200-HR was prepared and the column was washed with 3 volumes of column buffer (50 mM Tris, 100 mM NaCl, pH 7.5). Two hundred μl of plasma or serum were diluted with Tris buffer to a final concentration of 50 mM Tris at pH 7.5, centrifuged for 3 min at 8,000g and the supernatant was loaded on the column. Proteins were eluted in column buffer in 0.5 ml fractions. The early eluting fraction was named high molecular weight (HMW) fraction, the albumin containing fractions was collected after 3 ml and the last eluting fraction was named low molecular weight (LMW) fraction. The HMW and LMW fractions were acetone precipitated, the pellets were dissolved in column buffer and protein concentration was measured (by Nanodrop) prior to further analyses.