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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 staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.


Caroline May, Frederic Brosseron, Kathy Pfeiffer, Kristin Fuchs, Helmut E Meyer, Barbara Sitek, Katrin Marcus. Proteome Analysis with Classical 2D-PAGE. Methods in molecular biology (Clifton, N.J.). 2021;2228:53-62

PMID: 33950483

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