High Density Gel Electrophoresis

High-density gels can be synthesized having dimensions of the same order of magnitude as the molecular dimensions of protein molecules. In such gels, small molecules would migrate rapidly through the matrix unrestrained whereas larger, or more asymmetric molecules, would be retained and, thus, a separation could be achieved based on size-exclusion principles. This procedure would improve resolution significantly as the basis of molecular discrimination would depend on the widely differing shapes of the protein molecules.

Originally, high-density gels were synthesized from hydrolyzed starch but due to opacity their use for quantitative work was very limited. Modern high-density gels are prepared from polyacrylamide but as they are unstable, they are not commercially available and need to be prepared as required. Thee acrylamide content can range from3 to 30% and cross-linked with N, N’-methylene-bis acrylamide.

Figure 15. A Disk Separation of a Series of Proteins

Polymerization is initiated by the use of peroxides or by photochemical activation. The gels are usually formed in glass tubes each used for only one sample. The proteins, when stained, appear as a series of disks as depicted in figure 15. The technique can also be easily adapted for preparative purposes.