Difference gel electrophoresis. A single gel method for detecting changes in protein extracts

Abstract

We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.

Saturation fluorescence labeling of proteins for proteomic analyses

“…Differential In-Gel Electrophoresis; DIGE [8][9][10], or saturation labeling [8;11]) due to its potential for quantitative accuracy and sensitivity. The advantages of this approach include the opportunity for multiplexing samples and inclusion of internal standards, the potential for detecting low abundance proteins, the ability to realize relative and absolute linear quantification (once the protein is identified), and the potential for real-time monitoring of electrophoresis.…”

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…The DIGE makes use of fluorescent cyanine dyes, such as Cy3 and Cy5 [74][75][76][77] that differ in their wavelengths. They are used to label differentially two or more cell situations prior to 2-DE.…”

Two-dimensional gel electrophoresis in bacterial proteomics

“…One of the most notable advancements in proteome analysis made over recent years has been the development of an approach known as 2-D differential gel electrophoresis (2D-DIGE) (Unlü et al, 1997). This technique is an invaluable approach for analyzing global changes in protein expression patterns within a certain system (e.g.…”

“…by covalently labeling two or three different protein samples with different fluorescent dyes that have distinct excitation and emission characteristics, they may be mixed and analyzed together on the same 2-D gel ( Fig. 1) (Unlü et al, 1997;Marouga et al, 2005). Gels are visualized using a commercial fluorescent gel scanner/densitometer to quantify the fluorescence intensity, which is proportional to the amount of (labeled) protein present in each spot.…”