The development of the DIGE system: 2D fluorescence difference gel analysis technology

Abstract

Two-dimensional (2D) gel electrophoresis is a powerful technique enabling simultaneous visualization of relatively large portions of the proteome. However, the well documented issues of variation and lack of sensitivity and quantitative capabilities of existing labeling reagents, has limited the use of this technique as a quantitative tool. Two-dimensional difference gel electrophoresis (2D DIGE) builds on this technique by adding a highly accurate quantitative dimension. 2D DIGE enables multiple protein extracts to be separated on the same 2D gel. This is made possible by labeling of each extract using spectrally resolvable, size and charge-matched fluorescent dyes known as CyDye DIGE fluors. 2D DIGE involves use of a reference sample, known as an internal standard, which comprises equal amounts of all biological samples in the experiment. Including the internal standard on each gel in the experiment with the individual biological samples means that the abundance of each protein spot on a gel can be measured relative (i.e. as a ratio) to its corresponding spot in the internal standard present on the same gel. Ettan DIGE is the system of technologies that has been optimized to fully benefit from the advantages provided by 2D DIGE.

Proteomics of spermatogenesis: from protein lists to understanding the regulation of male fertility and infertility

“…For example, proteins without lysine cannot be labelled, and they require special equipment for visualisation. Additionally, these fluorophores are very expensive; 63,64 hence, this technique is not widely used.…”

Inhibitory activity of blasticidin A, a strong aflatoxin production inhibitor, on protein synthesis of yeast: selective inhibition of aflatoxin production by protein synthesis inhibitors

“…10 Our attempt to obtain BcA-resistant strains by transformation of S. cerevisiae with cDNA or genomic libraries has not afforded critical candidate genes for investigating a target molecule (data not shown). Therefore, analysis of the yeast proteome by two-dimensional differential gel electrophoresis (2D-DIGE) 11 was performed. There are many works on proteome of S. cerevisiae 12 and alterations in protein abundance levels can provide clues about the mechanism of action of various drugs.…”

Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state

“…After electrophoresis and scanning on an imager, integrated software can be used to co-detect, locate and analyze protein spots, followed by assigning statistical confidence to each and every difference via a differential analysis algorithm and thereby avoid gel-to-gel variations. For instance, differences as little as 10% can be routinely detected with >95% statistical confidence [13].…”