Two Dimensional Gel Electrophoresis.

Two-dimensional electrophoresis (2-D electrophoresis), as the name implies is a technique where a mixture is resolved by employing two different separating systems.  The first system, for example, could be achieved by isoelectric focussing and the second by a separation process based on the different masses of each protein. The separations are carried out on a square plate and initially the sample is placed in one corner of the plate and the solute migration carried out electrophoretically along one side of the square. The solutes will then arrayed along the edge of the square in order of their isoelectric points (i.e. their pI’s).

Before separating the proteins further on the basis of mass, the spots are treated with sodium dodecyl sulphate, which denatures the proteins (i.e. destroys their three dimensional structure and forms them into straight chains). Now, the unfolded length of a protein is roughly proportional to its mass so it attaches (by means of dispersion forces) a number of sodium dodecyl sulphate molecules that will also be proportional to its mass. It follows that, as the sodium dodecyl sulphate molecules are negatively charged, all the proteins will have approximately the same mass to charge ratio. Now, as a result of the initial isoelectric focussing step, the solutes are all situated at their pI’s and consequently cayy no charge. Nevertheless, as a result of the sodium dodecyl sulphate treatment they will now have a charge and can be caused to migrate over a given potential gradient. The smaller molecules at the front of the group of the group and the larger molecules at the rear of the group.

Figure 14. The 2 Dimensional Electrophoretic Separation of Some Proteins

The potential gradient is now, however, applied normal to the previous electric field. As the charge-to-mass ratio is approximately the same for all the solutes, their differential movement will be controlled by frictional forces (i.e. viscous drag) only; the larger molecules being retarded to a greater extent than the smaller molecules. The gel now acts like a molecular sieve and the contents of each spot separated on the same basis as that effective in exclusion chromatography. An example of a 2-dimensional gel electrophoretic separation of some proteins is shown in figure 14.