Large Gel Two-Dimensional Electrophoresis: Improving Recovery of Cellular Proteome

Abstract

One of the goals of expression proteomics is to display and analyze all the proteins in a particular proteome. Cells are thought to comprise tens of thousands of proteins expressed in a dynamic range of 1-10 5 or 10 6. Low recovery of cellular proteome leads to a gross loss of important proteins. Thus, proteomics demands a powerful technology that separates complex mixture of proteins including low abundant ones. In the case of two-dimensional gel electrophoresis (2- DE), enlargement of the gel size appears a straightforward and effective strategy for improving the recovery of cellular proteins. Multiple narrow pH range immobilized pH gradients (nrIPGs) and long isoelectric focusing (IEF) gels afford improved separation of proteins in the first dimension according to isoelectric point. In addition, multiple long SDSPAGE gels of different polyacrylamide concentrations provide a tool to improve the resolution of the second dimension according to molecular weight. Recent data suggest that 2-DE with large gels can display more than 11,000 protein spots expressed in a 1-10 5 dynamic range in cells. Keywords: proteome, two-dimensional gel electrophoresis, dynamic range, low abundance, resolution of separation, recovery