Concentration and analysis of proteins by a discontinuous pH gradient formed by isoelectric focusing in polyacrylamide gel

Abstract 1. 1. This paper describes a method for concentrating proteins before sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). 2. 2. The procedure is based on gel electrofocusing and the main modifications are that (i) acrylamide gels were cast into 150 × 2.5 mm glass tubes to a height of 15−18 mm, in the remainder of the tube an argininc solution (20 mM arginine. 10% glycerol, 5% 2-mercaptoethanol, pH 10.5) containing the protein to be concentrated was placed, (ii) the pH in the gels was maintained by a mixture of Ampholine pH range 3.5−5.0 and 9−11. 3. 3. Bovine serum albumin (protein load 4μg), ribonuclease (10μg) ovalbumin (10μg), chymotrypsinogen A (10μg) and hemoglobin (10μg) were concentrated for 2−8 hours at 100 V and 0−5 C with recoveries of about 83%. 4. 4. The gels containing the concentrated protein were loaded onto a vertical gel slab and analyzed as a function of molecular weight by SDS-PAGE.