Two-dimensional difference gel electrophoresis

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.

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

“…It represents a highly accurate quantitative technique that enables multiple protein samples to be separated in parallel on the same 2-DE gel, thereby greatly reducing the introduction of potential artifacts due to gel-to-gel variations [28]. …”

“…The method was previously validated in cardiac tissue and resulted in two distinct subproteomes by partitioning soluble and membrane-associated proteins into aqueous and detergent phases, respectively [27]. Here, 2-D fluorescence DIGE analysis [28] was used to compare the young adult versus the senescent rat gastrocnemius muscle. Following the gel electrophoretic separation of the aqueous and the detergent-extracted fraction from skeletal muscle homogenates, proteins of interest with a changed abundance were identified by MALDI-ToF MS, MALDI-ToF/ToF MS and ESI LC-MS/ MS technology.…”

Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse

“…Labeling was carried out with 200 pmol of Cy3 fluor dye per 25 lg protein [26]. An internal pooled standard of every sample was prepared with the Cy5 fluor.…”

“…Proteomic profiling of Dp427-deficient leg and diaphragm muscles has revealed drastic changes in the expression levels of adenylate kinase, the luminal Ca 2+ -binding protein calsequestrin (CSQ), the cytosolic Ca 2+ -binding protein regucalcin, and the cardiovascular heat shock protein (cvHsp), as well as a large number of metabolic and contractile muscle proteins, as recently reviewed by Lewis et al [2]. Here, we have used fluorescence difference in-gel electrophoresis (DIGE), one of the most powerful comparable techniques available in modern biochemistry [26], to study potential changes in protein expression levels in Dp427-deficient versus normal EOM preparations. The identification of only moderate changes in a few select proteins agrees with the notion of a naturally protected phenotype.…”

Native‐DIGE: A new look at the mitochondrial membrane proteome

“…With this technique, samples differentially labeled with spectrally resolvable fluorescent cyanine dyes Cy2, Cy3 and Cy5 (GE Healthcare) can be loaded onto the same gel, thereby reducing the total number of gels. DIGE can reliably detect the fold-change minimum of 1.15-fold in abundance of a protein, over a >10 000-fold protein concentration range [15]. Use of common internal standard (pool of all samples to be compared) on all gels minimizes gel to gel variation, ensures accurate normalization and allows convenient grouping of multivariate samples, though resolved on different gels.…”