Difference Gel Electrophoresis (DIGE)

Abstract

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

Alban A, David SO, Bjorkesten L et al. (2003) A novel experimental design for comparative two‐dimensional gel analysis: two‐dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3: 36–44.

Dani D and Dencher NA (2008) Native‐DIGE: a new look at the mitochondrial membrane proteome. Biotechnological Journal 3: 817–822.

Fodor IK, Nelson DO, Alegria‐Hartman M et al. (2005) Statistical challenges in the analysis of two‐dimensional difference gel electrophoresis experiments using DeCyder. Bioinformatics 21: 3733–3740.

Fu CX, Hu J, Liu T et al. (2008) Quantitative analysis of redox‐sensitive proteome with DIGE and ICAT. Journal of Proteome Research 7: 3789–3802.

GE Healthcare (2005) Ettan DIGE System User Manual. 18‐1173‐17AB.

GE Healthcare (2006) Amersham CyDye DIGE Fluor Labelling Kit for Scare Samples. 25‐8009‐83PL, Rev C.

GE Healthcare (2008) CyDye DIGE Fluors (minimal dyes) for Ettan DIGE. RPK0272PL Rev E4/2008.

Heinemeyer J, Scheibe B, Schmitz UK and Braun H‐P (2009) Blue native DIGE as a tool for comparative analyses of protein complexes. Journal of Proteomics 72: 539–544.

Hrebicek T, Duerrschmid K, Auer N, Bayer K and Rizzi A (2007) Effect of CyDye minimum labeling in differential gel electrophoresis on the reliability of protein identification. Electrophoresis 28: 1161–1169.

Jorrin JV, Maldonado AM and Castillejo MA (2007) Plant proteome analysis: a 2006 update. Proteomics 7: 2947–2962.

Kang Y, Techanukul T, Mantalaris A and Nagy JM (2009) Comparison of three commercially available DIGE analysis software packages: minimal user intervention in gel‐based proteomics. Journal of Proteome Research 8: 1077–1084.

Karp NA, Kreil DP and Lilley KS (2004) Determining a significant change in protein expression with DeCyder during a pair‐wise comparison using two‐dimensional difference gel electrophoresis. Proteomics 4: 1421–1432.

Karp NA and Lilley KS (2009) Investigating sample pooling strategies for DIGE experiments to address biological variability. Proteomics 9: 388–397.

Lilley KS, Razzaq A and Dupree P (2002) Two‐dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Current Opinion in Chemical Biology 6: 46–50.

Miller I, Crawford J and Gianazza E (2006) Protein stains for proteomic applications: which, when, why? Proteomics 6: 5385–5408.

Shaw J, Rowlinson R, Nickson J et al. (2003) Evaluation of saturation labelling two‐dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3: 1181–1195.

Shaw MM and Riederer BM (2003) Sample preparation for two‐dimensional gel electrophoresis. Proteomics 3: 1408–1417.

Timms JF and Cramer R (2008) Difference gel electrophoresis. Proteomics 8: 4886–4897.

Unlu M, Morgan ME and Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18: 2071–2077.

Viswanathan S, Unlu M and Minden JS (2006) Two‐dimensional difference gel electrophoresis. Nature Protocols 1: 1351–1358.

Westermeier R and Marouga R (2005) Protein detection methods in proteomics research. Bioscience Reports 25: 19–32.

Wilkins MR, Appel RD, Van Eyk JE et al. (2006) Guidelines for the next 10 years of proteomics. Proteomics 6: 4–8.

Wu WW, Wang GH, Baek SJ and Shen RF (2006) Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel‐ or LC‐MALDI TOF/TOF. Journal of Proteome Research 5: 651–658.

Further Reading

GE Healthcare (2008) DeCyder 2D software, Version 7.0, User manual, 28‐9414‐47 AA 10/2008.

GE Healthcare (2008) DeCyder Extended Data Analysis, Version 7.0, 28‐9414‐44 AA 10/2008.

Jung ME and Kim W‐J (2006) Practical syntheses of dyes for difference gel electrophoresis. Bioorganic & Medical Chemistry 14: 92–97.

Tannu NS and Hemby SE (2006) Two‐dimensional fluorescence difference gel electrophoresis for comparative proteomic profiling. Nature Protocols 1: 1732–1742.

Tannu NS and Hemby SE (2006) Quantitation in two‐dimensional fluorescence difference gel electrophoresis: effect of protein fixation. Electrophoresis 27: 2011–2015.

Westermeier R, Naven T and Höpker H‐R (2008) Proteomics in Practice: A Guide to Successful Experimental Design. Weinheim: Wiley‐VCH.

Web Links

DIGE guide at GE Healthcare http://www4.gelifesciences.com/aptrix/upp01077.nsf/Content/2d_electrophoresis∼new_to_2d_dige

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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]