Difference Gel Electrophoresis (DIGE)

Irene Granlund, Swedish University of Agricultural Sciences, Umeå, Sweden
Michael Hall, Umeå University, Umeå, Sweden
Wolfgang P Schröder, Umeå University, Umeå, Sweden

Published online: December 2009

DOI: 10.1002/9780470015902.a0021881

One of the largest challenges in proteomics today is to be able to quantify the composition and amount of proteins found in a specific cell or tissue at a defined time point. Difference gel electrophoresis (DIGE) is a gel electrophoresis-based technique for protein quantification in complex mixtures. In DIGE the high resolution of two-dimensional gel electrophoresis is combined with the excellent dynamic range obtained by fluorescent tag labelling of protein samples. The output of DIGE experiments provides information about how many proteins display changed expression levels on a specific treatment. In addition, proteins of interest can be excised and identified with conventional mass spectrometry techniques and further analysed by other biochemical methods.

Key concepts:

  • DIGE is a gel-based technique for relative protein quantification in complex protein samples.
  • Experimental design, sample preparation and data analysis are all critical steps of a DIGE experiment.
  • Labelling of protein samples is performed by either a minimal or saturation labelling procedure.
  • Use of an internal standard minimizes gel to gel variation and provides increased power to the experiment.
  • For protein identification poststaining of gels is necessary when a minimal labelling procedure has been used.
  • DIGE can also be used for other applications such as native gel electrophoresis of protein complexes.
  • The DIGE technique is constantly being improved and continues to be an important method in functional proteomics.

Keywords: DIGE; 2D-PAGE; CyDye; protein expression; fluorescence

Figure 1. Work flow of a DIGE experiment, showing the important steps in the technique. (1) The experimental design where the evaluation of the possible gain of the experiment is made and the decision of which samples will be compared, how many biological and technical replicates are needed, how many dyes will be used and what type of labelling procedure will be applied (minimal or saturation). (2) Labelling of the samples with the desired labelling method. (3) Separation of the samples with a suitable separation technique (usually IEF/SDS-PAGE). (4) Scanning of the gels with a multiwavelength scanner at high resolution. (5) Image analysis with one or more software packages designed for DIGE. (6) Statistical analysis and selection of spots exhibiting significant changes in relative average abundance. (7) Poststaining, matching, picking of the proteins of interest and tryptic digestion. (8) Identification of the protein spots by mass spectrometry methods, for example MALDI (matrix-assisted laser desorption ionization)-TOF (time of flight)-TOF or LC (liquid chromatography)-MS/MS.
Figure 2. Labelling procedures for DIGE experiments. The left side shows the minimal labelling procedure where three dyes are used. In the internal standard the same amount of all the samples in the experiment are pooled together and labelled with Cy2. The samples to be analysed are labelled with the other dyes (Cy3 and Cy5) in a reciprocal fashion (dye-swap). The right side shows the saturation labelling procedure where only two dyes are used, were TCEP stands for tris-(2-carboxyethyl) phosphine hydrochloride, a special reductant for cysteins used with this dye.
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 References
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 Further Reading
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    other GE Healthcare (2008) DeCyder Extended Data Analysis, Version 7.0, 28-9414-44 AA 10/2008.
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    Tannu NS and Hemby SE (2006) Quantitation in two-dimensional fluorescence difference gel electrophoresis: effect of protein fixation. Electrophoresis 27: 2011–2015.
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 Web Links
    ePath DIGE guide at GE Healthcare http://www4.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis~new_to_2d_dige

Related Sub-Topics:

Basic Cell Biology, Protein, RNA and DNA | Techniques in Cell Biology | Protein Structure | Biochemical and Biophysical Techniques | Proteins: Structure, Function, Metabolism
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Article Title: Difference Gel Electrophoresis (DIGE)
 
How to Cite close
Granlund, Irene, Hall, Michael, and Schröder, Wolfgang P(Dec 2009) Difference Gel Electrophoresis (DIGE). In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021881]