Two-dimensional electrophoresis of proteins with a different approach to isoelectric focusing

Abstract

A simple method for the two-dimensional analysis of proteins was developed based on direct extraction of a protein sample from dried/precipitated material into isoelectric focusing (IEF) gel solution. This sample solution is equally useful for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and two-dimensional electrophoresis. For the former method this sample solution is applied directly to the gel. For the latter, sample solution containing a desired amount of proteins is mixed with IEF gel solution followed by polymerization. The method thus allows solubilization in volumes as large as the total IEF gel volume, uses no salt and SDS in sample solublization and needs no boiling. It is thus simple and can be used for the analysis of samples containing very small amounts of proteins and/or with low radioactive incorporation without involving concentration procedures that result in further losses of proteins. The presence of proteins within the polymerized gel prior to the run removes any doubt regarding their entry into the gel, and an extended pH gradient results in perfect resolution.