State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research |
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| Authors: | Carrette Odile Burkhard Pierre R Sanchez Jean-Charles Hochstrasser Denis F |
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| Institution: | Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, Faculty of Medicine, Geneva University, 1 rue Michel Servet CH-1211 Geneva 4, Switzerland. |
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| Abstract: | Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomics technologies. Based on two distinct procedures, it combines isoelectric focusing (IEF), which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. At present, 2D-PAGE is capable of simultaneously detecting and quantifying up to several thousand protein spots in the same gel image. Here we provide comprehensive step-by-step instructions for the application of a standardized 2D-PAGE protocol to a sample of human plasma or cerebrospinal fluid (CSF). The method can be easily adapted to any type of sample. This four-day protocol provides detailed information on how to apply complex biological fluids to an immobilized dry strip gel, cast home-made gradient acrylamide gels, run the gels, and perform standard staining methods. A troubleshooting guide is also included. |
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