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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.

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Abbreviations

CyDye DIGE fluor Cy2 minimal dye:

(3-(4-Carboxymethyl)phenylmethyl)-3′-ethyloxacarbocyanine halide N-hydroxysuccinimidyl ester)

CyDye DIGE fluor Cy3 minimal dye:

(1-(5-Carboxypentyl)-1′-propylindocarbocyanine halide N-hydroxysuccinimidyl ester)

CyDye DIGE fluor Cy5 minimal dye:

(1-(5-Carboxypentyl)-1′-methylindodicarbocyanine halide N-hydroxysuccinimidyl ester)

CyDye DIGE fluor Cy3 maleimide dye:

1-(6-([2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino)-6-oxohexyl)-2-[(1E,3E)-3-(1-ethyl-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)prop-1-enyl]-3,3-dimethyl-3H-indolium

CyDye DIGE fluor Cy5 maleimide dye:

1-(6-([2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino)-6-oxohexyl)-3,3-dimethyl-2-[(1E,3E,5E)-5-(1,3,3-trimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-3H-indolium

DIGE:

Difference gel electrophoresis

DIA:

Differential in-gel analysis

BVA:

Biological variation analysis

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Correspondence to Rita Marouga.

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Cy, CyDye, DeCyder, Ettan, ImageMaster and Typhoon are trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc.

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Marouga, R., David, S. & Hawkins, E. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal Bioanal Chem 382, 669–678 (2005). https://doi.org/10.1007/s00216-005-3126-3

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  • DOI: https://doi.org/10.1007/s00216-005-3126-3

Keywords

  • 2D DIGE
  • CyDye DIGE fluors
  • DeCyder differential analysis software
  • Fluorescent labeling
  • Proteomics