The Method of Two-Dimensional Differential Gel Electrophoresis and Its Application in Proteomics
Department of Genetics and Plant Breeding, Crop Research Institute, Prague
Two-dimensional differential gel electrophoresis (2-D DIGE) is a modification of the well-known two-dimensional method (2-DE). The new method enables separation of two protein samples on one 2-D gel thus avoiding problems associated with reproducibility of 2-D gels and makes it possible to compare different samples. The only extra step in 2-D DIGE is sample labelling with cyanine fluorescent dyes (CyDyes). Two types of labelling are used: with succinimidyl esters and with maleimide derivatives of the dyes to label lysine and cysteine residues, respectively. For detection and quantification of protein spots in gels, a special fluorescent scanner or a CCD camera are required. From densitometric analysis of 2-D DIGE gels, sets of multidimensional data are obtained, which are then analysed by statistical methods. Protein spots can be cut from 2-D DIGE gels and further analysed by MS. The 2-D DIGE method affords much better quantification of proteins in samples. The separation of two samples at a time leads to a large reduction in the used amount of 2-D gels.
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