Isoelectric Focussing Essay
Isoelectric Focussing The method of separating proteins according to their isoelectric points in a pH gradient is called isoelectric focusing. This technique was discovered by H.Svensson in Sweden. This method has a high resolution power because ordinary paper electrophoresis resolves plasma proteins into six bands where as isoelectric focusing resolves it into 40 bands. In conventional electrophoresis the pH between anode and cathode is constant and the positively charged ions migrate toward the cathode and the negative ions migrate toward anode. But in isoelectric focusing, a stable pH gradient is arranged. The pH gradually increases from anode to cathode. When a protein is introduced at a pH which is lower than its isoionic point, it will possess a net positive charge and will migrate in the direction of the cathode.
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.
University/College: University of California
Type of paper: Thesis/Dissertation Chapter
Date: 3 June 2017
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