The Use of Difference In-Gel Electrophoresis for Quantitation of Protein Expression

Summary

Two-dimensional difference in-gel electrophoresis (2D-DIGE) is a modified 2D electrophoresis (2DE) technique that enables comparison of two (or three) proteomes simultaneously on the same gel. The different protein samples to be compared are covalently tagged with spectrally different fluorescent dyes that are designed to have no effect on the relative migration of proteins during isoelectric focusing or molecular mass separation during electrophoresis. The “spot maps” generated from the dye scans for each of the dyes are then superimposed to discern the expression pattern of the proteome samples being compared. Proteins that do not change in expression are seen as spots with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. The fluorescent dyes used in DIGE are cyanine flours and matched in respect of molecular weight. Two different dye chemistries are available enabling fluorescent tagging of as low as 5 μ g of proteins to get the analysis of the regulation of the proteome. Furthermore, DIGE is a sensitive technique, capable of detecting as little as 0.5 fmol of protein, and this detection system is linear over a >10,000-fold concentration range.