Three-dimensional electrophoresis for quantitative profiling of complex proteomes

Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post- translational modifications (PTMs) detection. A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.