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تسجيل

Two-Dimensional Polyacrylamide Gel Electrophoresis

المؤلف:  Wilson, K., Hofmann, A., Walker, J. M., & Clokie, S. (Eds.)

المصدر:  Wilson and Walkers Principles and Techniques of Biochemistry and Molecular Biology

الجزء والصفحة:  8th E , P233-234

2026-04-14

467

 This technique combines the technique of IEF (first dimension), which separates proteins in a mixture according to charge (pI), with the size separation technique of SDS–PAGE (second dimension). The combination of these two techniques to give two-dimensional (2D) PAGE provides a highly sophisticated analytical method for analysing protein mixtures. To maximise separation, most workers use large format 2D gels (20 cm × 20 cm), although the minigel system can be used to provide useful separation in some cases. For large-format gels, the first dimension (isoelectric focus sing) is carried out in an acrylamide gel that has been cast on a plastic strip (18 cm × 3 mm wide). The gel contains ampholytes (for forming the pH gradient) together with 8 M urea and a non-ionic detergent, both of which denature and maintain the solubility of the proteins being analysed. The denatured proteins therefore separate in this gel according to their isoelectric points. The IEF strip is then incubated in a sample buffer containing SDS (which associates with the denatured proteins) and then placed between the glass plates of, and on top of, a previously prepared 10% SDS–PAGE gel. Electrophoresis is commenced and the SDS-bound proteins run into the gel and separate according to size. The IEF gels are provided as dried strips and need rehydrating overnight. The first-dimension IEF run takes 6–8 h, the equilibration step with SDS sample buffer about 15 min, and then the SDS–PAGE step takes about 5 h. A typical 2D gel is shown in Figure 1. Using this method, one can routinely resolve between 1000 and 3000 proteins from a cell or tissue extract, and in some cases workers have reported the separation of between 5000 and 10 000 proteins.

Fig1. A typical two-dimensional gel. The sample applied was 100 µg of total protein extracted from a normal dog heart ventricle. The first dimension was carried out using a pH 4–7 isoelectric focussing gel. The second dimension was a 12% SDS–PAGE vertical slab gel. The pattern was visualised by silver staining. (Courtesy of Monique Heinke and Dr Mike Dunn, Division of Cardiothoracic Surgery, Imperial College School of Medicine, Heart Science Centre, Harefi eld, UK.)