Abstract
Two-dimensional difference gel electrophoresis (2D DIGE) is a modified 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 fluorescent 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 fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence 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 >10,000-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 2–3 d to complete.
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JM has a conflict of interest with respect to DIGE and the ProteoHook protein clean up kit. These methods were developed in the Minden lab and licensed to Amersham and Proteopure, respectively. JM receives royalties for DIGE from Amersham and options from Proteopure. SV is an employee of Proteopure.
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Supplementary Video 1
DIGE movie. Shown here is a two-frame, looping movie comparing different mouse cell extracts. (MOV 195 kb)
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Viswanathan, S., Ünlü, M. & Minden, J. Two-dimensional difference gel electrophoresis. Nat Protoc 1, 1351–1358 (2006). https://doi.org/10.1038/nprot.2006.234
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DOI: https://doi.org/10.1038/nprot.2006.234
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