Two-dimensional gel electrophoresis


Two-dimensional gel electrophoresis (2-D electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique separate proteins in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW). In this way, complex mixtures consisted of thousands of different proteins can be resolved and the relative amount of each protein can be determined.

The procedure involves placing the sample in gel with a pH gradient, and applying a potential difference across it. In the electrical field, the protein migrates a long the pH gradient, until it carries no overall charge. This location of the protein in the gel constitutes the apparent pI of the protein.

There are two alternatives methods to create the pH gradient - carrier ampholites and immobilized pH gradient (IPG) gels. In the Maiman Institute for Proteome Research the IEF is performed with commercial IPGs for highly reproducible results.

The IEF is the most critical step of the 2-D electrophoresis process. The proteins must be solubilize without charged detergents, usually in high concentrated urea solution, reducing agents and chaotrophs. To obtain high quality data it is essential to achieve low ionic strength conditions before the IEF it self. Since different types of samples differ in their ion content, it is necessary to adjust the IEF buffer and the electrical profile to each type of sample.

The separation in the second dimension by molecular size is performed in slab SDS- PAGE. Twelve parallel gels can be separated in a fixed temperature to minimize the separation variations between individual gels.


The Maiman Institute for Proteome Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel