Due to the presence of pH gradient, the net charge of the molecule changes due to ionization as it moves forward. When the protein encounters a pH where its net charge is zero, it will stop migrating. This is the isoelectric point of the protein. Every protein present in the mixture will migrate to its isoelectric point and stops its migration there itself. Thus, once a final stable focusing is reached, the resolution will be retained for a long time. Enzyme proteins resolved by IEF are then separated in a second dimension based on their molecular weight.
To conduct this, IEF gel is extruded from the tube and placed lengthwise on a slab gel of polyacrylamide saturated with SDS. When an electric current is applied, the enzymeproteins migrate from the IEF gel into the SDS gel and then get separated according to their mass. This method helps in excellent separation of cellular enzyme-proteins. Uses: The two dimensional gel electrophoresis is used in developmental biochemistry to monitor the increase or decrease in the intensity of a spot representing as specific protein as a function of cell growth. It is a standard method of judging protein purity.