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Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome
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Proteome analysis with classical 2D-PAGE

Caroline May1, Frederic Brosseron, Kathy Pfeiffer

  • 1Department of Medical Proteomics/Bionalaytics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany. caroline.may@rub.de

Methods in Molecular Biology (Clifton, N.J.)
|June 6, 2012
PubMed

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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Two-dimensional Gel Electrophoresis

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