Gradient gel electrophoresis

 

By continuously changing the acrylamide concentration in the polymerization solution, a pore gradient gel is obtained. It can be used to determine the molecular diameter of proteins in their native state.

When the acrylamide concentration and the degree of crosslinking are fixed high enough in the small pore area, the protein molecules reach a point where, because of their size, they are trapped in the tight gel matrix. Since the speed of migration of the individual protein molecules depends on their charge, the electrophoresis must be carried out long enough so that the molecule with the lowest net charge also reaches its end point.

 

The are various way of preparing gels with linear or exponential pore gradients. All are based on the same principle: two polymerization solutions with different acrylamide concentrations are prepared. During casting, the dilute solution is continuously mixed with the concentrated solution so that the concentration in the casting mold decreases from bottom to top.

The density of the highly concentrated solution is increased with glycerol or sucrose so that the layers in the molds do not mix. In principle a concentration gradient is poured. The mixing of the less dense dilute solution with the viscous solution takes place in the mixing chamber using a magnetic stirrer bar.

A discontinuity only exists at the front.

The determination of molecular weights in this manner can be problematic, since different proteins have different tertiary structures. Structural proteins cannot be compared with globular proteins.

When single gradient gels are cast, the solution is poured into the top of the mold. When several gels are cast simultaneously, the solutions are injected from the bottom into multiple molds. In this case, the solutions in the mixing chamber and the reservoir are interchanged.

If the mixing chamber is left open at the top, the principle of communicating vases is valid: so that the height of both fluids stays equal, half as much of the dilute solution flows in as of solution flowing out of the mixing chamber. A linear gradient is thus formed. A compensating stick in the reservoir compensates the volume of the stirrer bar and the difference in the densities of both solutions