Abstract
This chapter describes the basics, applications, and limitations of two-dimensional gel electrophoresis (2DE) and two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of distinct proteomes. We also propose a basic protocol for 2D-DIGE, technique that allows the analysis of paired protein extracts, which are labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed with a Cy2-labeled standard extract on the same 2DE gels. Scanning the gels at wavelengths specific for each dye allows direct overlay the two different proteomes and the differences in abundance of specific protein spots can be determined.
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References
Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF et al (1996) Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 13:19–50
O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Görg A, Postel W, Weser J, Gunther S, Strahler JR, Hanash SM et al (1987) Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8:122–124
Görg A, Postel W, Günther S (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9:531–546
López JL (2007) Two-dimensional electrophoresis in proteome expression analysis. J Chromatogr B Analyt Technol Biomed Life Sci 849:190–202
Thomas A, Lenglet S, Chaurand P, Deglon J, Mangin P, Mach F et al (2011) Mass spectrometry for the evaluation of cardiovascular diseases based on proteomics and lipidomics. Thromb Haemost 106:20–33
Penque D (2009) Two-dimensional gel electrophoresis and mass spectrometry for biomarker discovery. Proteomics Clin Appl 3:155–172
Oliveira BM, Coorssen JR, Martins-de-Souza D (2014) 2DE: the phoenix of proteomics. J Proteomics 104:140–150
Unlü M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077
Knowles MR, Cervino S, Skynner HA, Hunt SP, de Felipe C, Salim K et al (2003) Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis. Proteomics 3:1162–1171
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (1996) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
Acknowledgments
Dr. Adriano Aquino and Prof. Daniel Martins-de-Souza are funded by FAPESP (São Paulo Research Foundation), grants 15/09159-8 and 13/08711-3.
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Aquino, A., Guest, P.C., Martins-de-Souza, D. (2017). Simultaneous Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Analysis of Two Distinct Proteomes. In: Guest, P.C. (eds) Multiplex Biomarker Techniques. Methods in Molecular Biology, vol 1546. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-6730-8_17
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DOI: https://doi.org/10.1007/978-1-4939-6730-8_17
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