| 2D Gel Electrophoresis to Identify Cellular Proteins |
While computer-based methods are powerful, they can only predict the function of proteins for which some information is already available. How do we understand the proteins that we don't already know about? This requires experimental approaches.
One way to identify proteins is to extract all the proteins from a sample of cells and separate them in a gel matrix, using a technique called polyacrylamide gel electrophoresis (PAGE). The proteins are separated by size, with the smaller proteins moving faster through the gel than the larger proteins. After staining, a pattern of bands appears that corresponds to the proteins in the cell. However, this technique can only resolve a few hundred proteins, and cannot separate proteins of very similar size.
A modification of this procedure - called 2D gel electrophoresis - separates proteins into two dimensions, using two different characteristics. Proteins are separated in the first dimension by their isoelectric point (pI), the specific point at which the net charge of the protein is zero. These separated proteins, in a flat gel strip, are then placed on a standard polyacrylamide gel. Every protein band that was separated in the first dimension according to its isoelectric point is now separated in the second dimension by its size. The result is small spots, each representing a protein; even proteins of the same size will be resolved if they have a different isoelectric point. A good 2D gel can resolve one thousand to two thousand proteins, which appear, after staining, as dots in the gel (Fig. 4). This technique is useful when comparing two similar samples to find specific protein differences; for example, comparing the proteins in a tumor cell versus a normal cell. However, it can miss very small proteins or non-abundant proteins.