Reverse Isoelectric Focusing Procedure Resolves Charge Variants of Basic Proteins

Abstract

Several powerful methods exist which effectively resolve proteins based on their differences in overall charge. Among these, two-dimensional gel electrophoresis, a method combining isoelectric focusing with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, can resolve over 1000 proteins with isoelectric points in the range of pH 4-7. However, resolution of basic proteins is poor, even when the pH gradient is extended to a higher pH. Nonequilibrium pH gradient electrophoresis was developed as an alternative procedure to resolve basic proteins. However, samples must be dissolved in urea prior to analysis and this method requires 2000-8000 V-h for optimal resolution. In this report we demonstrate that a simple, rapid procedure, based on nonequilibrium pH gradient electrophoresis, can resolve the single charge variants of two fungal enzymes with isoelectric points greater than 8. This procedure utilizes ready-made materials and requires less than 500 V-h to complete (total run time is less than 2 h).