Protein profiling using two-dimensional difference gel electrophoresis (2-D DIGE)

2D-DIGE relies on pre-electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one gel. The dye-labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with spot matching between gels. Image analysis programs can then be used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable expression differences to be identified and quantified. This unit describes the DIGE procedure in terms of sample preparation from various types of cells, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples.