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Two-dimensional difference gel electrophoresis
Surya Viswanathan1,2, Mustafanl1,3 & Jonathan S Minden1
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1Department of Biological Science, Carnegie Mellon University, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA. 2Present address: Proteopure Incorporated, 900 William Pitt Way, Pittsburgh, Pennsylvania 15238, USA. 3Present address: Bogazici University, Department of Molecular Biology and Genetics, Bebek, 34342, Istanbul, Turkey. Correspondence should be addressed to J.M. ([email protected])
Published online 26 October 2006; doi:10.1038/nprot.2006.234
Two-dimensional difference gel electrophoresis (2D DIGE) is a modied form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color uorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as spots with a xed ratio of uorescent signals, whereas proteins that differ between the samples have different uorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.5 fmol of protein, and protein differences down to 15%, over a 410,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 23 d to complete.
INTRODUCTIONThe central goals of proteomics include identifying protein changes that differentiate normal and diseased states in cells, tissues or organisms, and examining how protein changes correlate with developmental age and environment. The rst stage in comparative proteomics is to separate complex mixtures of protein into individual components; this is typically done using gel electrophoresis (at the whole protein level) or column chromatography (at the peptide level). Both of these separation schemes have advantages and disadvantages. We have focused on two-dimensional electrophoresis (2DE) because of its accessibility to most laboratories. This approach was described simultaneously by several groups in 1975 (refs. 13). Despite the substantial advances in the technology since its launch the most notable of which was the introduction of immobilized pH gradients in the rst dimension4,5 some of the more signicant...