Analytical isoelectric focusing using a high-voltage vertical slab polyacrylamide gel system

Abstract

A commercially supplied vertical slab electrophoresis system has been modified to permit electrofocusing of thin gels using electrical potentials of 3000 V and higher. Polyacrylamide gels (5.65% T, 2.65% C; 2.4–3.3% ( w v ) ampholytes; 0.35 mm thick × 98–105 mm long × 140 mm wide) were run under native and denaturing conditions. Accurate temperature regulation and atmospheric control were obtained by casting the gel between two glass plates, and then completely submerging the gel in the lower tank buffer. As many as 18 samples were loaded into wells at the top of each gel. Protein standards and mouse ascites fluid were focused on gels in the native state using a broad-range blend of commercial ampholytes from pH 3.5 to 10. Narrow-range pH ampholyte blends were also used: pH 2.5 to 6 under denaturing conditions resolving bovine calmodulins; pH 4 to 6 under a native condition for human plasma proteins including immunoglobulin G, fibronectin, and fibrinogen; pH 4 to 6 under denaturing conditions for myosin light chains; pH 6 to 9 under native conditions for human hemoglobins; and pH 9 to 11 under denaturing conditions to separate 30 S ribosomal subunit proteins. High-voltage vertical slab electrofocusing provides a means for rapid resolution of multiple protein samples using stable pH gradients. The method is especially valuable in ranges near pH 2.5 and pH 10.5 in which difficulties have previously been encountered with regard to atmospheric control and temperature regulation using conventional focusing techniques.