A two-dimensional gel elctrophoresis protocol has been developed which provides for a 1.5-to 3-fold increase in the resolution of proteins compared to other frequently used methods. The major variations from previous protocols include increased pore size in the isoelectric focusing gels; cholamidopropyldimethylhydroxypropanesulfonate, a zwitterionic detergent, replaces most of the Nonidet P-40, a nonionic detergent, in the isoelectric focusing gels; no equilibration step is employed between the first and second dimensional separation. The use of a stacking gel in the second dimension has been eliminated; a more efficient and evenly distributed cooling system has been designed for the molecular mass separation, allowing faster migration with higher current. Finally, the crosslinker diacrylylpiperazine is employed which improves protein separation and detection with ammoniacal silver staining. Silver-stained two-dimensional gel electrophoretograms of human plasma and hamster brain tissues and autoradiographs of rat liver cells are compared to the results obtained from previous methods.