Abstract
Classical two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal as well as saturation labeling.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rabilloud T, Chevallet M, Luche S et al (2010) Two-dimensional gel electrophoresis in proteomics: past, present and future. J Proteomics 73(11):2064–2077
Rabilloud T, Vaezzadeh AR, Potier N et al (2009) Power and limitations of electrophoretic separations in proteomics strategies. Mass Spectrom Rev 28:816–843
Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077
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
Tonge R, Shaw J, Middleton B et al (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377–396
Helling S, Schmitt E, Joppich C et al (2006) 2-D differential membrane proteome analysis of scarce protein samples. Proteomics 6:4506–4513
Sitek B, Luttges J, Marcus K et al (2005) Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma. Proteomics 5:2665–2679
Shaw J, Rowlinson R, Nickson J et al (2003) Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3:1181–1195
Dowsey AW, English J, Pennington K et al (2006) Examination of 2-DE in the Human Proteome Organisation Brain Proteome Project pilot studies with the new RAIN gel matching technique. Proteomics 6:5030–5047
Acknowledgements
This work was supported by the Bundesministerium für Bildung und Forschung (NGFN, FZ 01GS08143) as well as the European Regional Development Fund (ERDF) of the European Union and the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen (ParkChip, FZ 280381102).
Thanks to Dr. Jan Heise (NH DyeAGNOSTICS GmbH) for providing S-Dyes and G-Dyes.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
May, C., Brosseron, F., Chartowski, P., Meyer, H.E., Marcus, K. (2012). Differential Proteome Analysis Using 2D-DIGE. In: Marcus, K. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 893. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-885-6_6
Download citation
DOI: https://doi.org/10.1007/978-1-61779-885-6_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-884-9
Online ISBN: 978-1-61779-885-6
eBook Packages: Springer Protocols