Proteins vary considerably in their charges and, consequently, in their pI values (pH at which their charge is zero). Separating proteins by isoelectric focusing requires establishment of a pH gradient in an acrylamide gel matrix. The matrix’s pores are adjusted to be large to reduce the effect of sieving based on size. Molecules to be focused are applied to the gel with the pH gradient and an electric current is passed through it. Positively charged molecules, for example, move towards the negative electrode, but since they are traveling through a pH gradient, as they pass through it, they reach a region where their charge is zero and, at that point, they stop moving. They are at that point attracted to neither the positive nor the negative electrode and are thus “focused" at their pI. By using isoelectric focusing, it is possible to separate proteins whose pI values differ by as little as 0.01 units.
2-D Gel Electrophoresis
Both SDS-PAGE and isoelectric focusing are powerful techniques, but a clever combination of the two is a powerful tool of proteomics - the science of studying all of the proteins of a cell/tissue simultaneously. In 2D gel electrophoresis, an extract containing the proteins is first prepared. One might, for example, be studying the proteins of liver tissue. The liver cells are lysed and all of the proteins are collected into a sample. Next, the sample is subjected to isoelectric focusing as described earlier, to separate the proteins by their pI values. Next, as shown on the previous page, the isoelectric gel containing the separated proteins is rotated through 90º and placed on top of a regular polyacrylamide gel for SDS-PAGE analysis (to separate them based on size). The proteins in the isoelectric gel matrix are electrophoresed into the polyacrylamide gel and separation on the basis of size is performed. The product of this analysis is a 2D gel, in which proteins are sorted by both mass and charge.
The power of 2D gel electrophoresis is that virtually every protein in a cell can be separated and appear on the gel as a distinct spot. In the figure, spots in the upper left correspond to large positively charged proteins, whereas those in the lower right are small negatively charged ones. It is possible using high- throughput mass spectrometry analysis to identify every spot on a 2D gel. This is particularly powerful when one compares protein profiles between different tissues or between the samples of the same tissue treated or untreated with a particular drug. Comparison of a 2D separation of a non-cancerous tissue with a cancerous tissue of the same type provides a quick identification of proteins whose level of expression differs between them. Information such as this might be useful in designing treatments or in determining the mechanisms by which the cancer arose.