Difference Gel Electrophoresis: Application in Quantitative Proteomics Research

Separation of proteins from biological samples by two-dimensional polyacrylamide gel electrophoresis (2D -PAGE) followed by their identification by mass spectrometry is a widely used technique in proteomics. Two dimensional difference gel electrophoresis (2D-DIGE) is a relatively new technique in 2D-PAGE for multiplex quantitative analysis of the component proteins of related but different protein samples. In this technique, prior to electrophoresis, the protein samples are labeled with different fluorescent dyes that have distinct spectral characteristics. The labeled samples are subsequently combined and then subjected to gel electrophoresis. Fluorescent dyes are available for this purpose that are matched for charge and mass. This enables co-separation, co-detection and simultaneous quantitative estimation of the components of different protein samples on the same gel. Thus, DIGE is useful for multiplex analysis. Commercially available CyDye DIGE Fluor minimal dyes have been widely used in 2D-DIGE in order to study simultaneous expression of protein molecules from related but different samples. On the other hand, CyDye DIGE Fluor saturation dyes are more recently available dyes used for such purpose and are particularly useful for studying biological samples, which are available in limited quantity. The chemistry for labeling the protein samples with CyDye DIGE Fluor minimal dyes is different from that for labeling the protein samples with CyDye DIGE Fluor saturation dyes. The present article will discuss the advantages and disadvantages of these dyes as well as the application of DIGE in the rapidly growing area of quantitative proteomics research.