Abstract
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures, such as Coomassie, silver, or fluorescence staining. This chapter gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.
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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).
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May, C., Brosseron, F., Pfeiffer, K., Meyer, H.E., Marcus, K. (2012). Proteome Analysis with Classical 2D-PAGE. 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_3
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DOI: https://doi.org/10.1007/978-1-61779-885-6_3
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