Two-dimensional difference gel electrophoresis.

Abstract

The two-dimensional (2D) polyacrylamide gel-based approach to protein profiling has been successful because it is an accessible, inexpensive, and powerful tool for the analysis of global patterns of protein expression. All protein spots that are resolved and detected within the 104 to 105 dynamic range of gel capacity can be studied qualitatively and quantitatively in relation to each other, and viewed as a single image. Two-dimensional difference gel electrophoresis (DIGE) has strengthened the 2D platform by allowing the detection and quantitation of differences between samples resolved on the same gel, or across multiple gels, when linked by an internal standard. The technology is based on modification of protein extracts with fluorescent cyanine dyes, which have distinct excitation and emission spectra and are migration (charge and size) matched so that the same protein labeled with any of the dyes (Cy2, Cy3, Cy5) will migrate to the same position within a 2D gel, greatly enhancing reproducibility. It is becoming clear that each technology that is currently available for quantifying differential protein expression has its own weaknesses and strengths, and that multiplatform, integrated approaches will be necessary to provide the most complete analysis of any given proteome. We believe that DIGE is, and will remain in the future, a key front-end proteomic tool that will strongly complement other protein-profiling technologies.