Purification of human serum gamma globulins by hydrophobic interaction high-performance liquid chromatography

Fresh, whole human serum was fractionated on a Bio-Rad Protein Chromatography System, equipped with a Bio-Gel TSK Phenyl-5PW column, by utilizing a descending linear gradient of ammonium sulfate in 0.1 M sodium phosphate buffer, pH 7.0, at 0 degree C. Two major peaks were isolated corresponding to albumin and gamma globulin. The identity of these protein peaks was substantiated by chromatography of an albumin-gamma globulin standard mixture. The purity of the individual fractions was verified by high-performance size exclusion chromatography (HPSEC) on either a Bio-Sil TSK-250 or a Bio-Gel TSK-40 column. The applicability of these HPSEC columns to the molecular weight characterization of the Bio-Gel TSK Phenyl-5PW column fractions was compared. Typically, the Bio-Gel TSK Phenyl-5PW column (75 X 7.5 mm I.D.) was used to purify gamma globulin from 100 microliter of plasma. This corresponded to approx. 1.5-2.0 mg of the globin fraction. Unidentified contaminants in this fraction had molecular weights of approx. 1000-3000 daltons and 260 000-300 000 daltons.