Lecture notes on Electrophoresis

Electrophoresis is a separation technique, which depends on the ability of charged molecules to migrate when placed in an electric field. Electrophoresis has been developed into a most potent technique for the isolation and identification of a wide variety of biological materials like proteins, nucleotides, peptides, etc. The principle of electrophoresis was given by the Russian Physiologist Alexander Reuss in 1807. if a mixture of two or more different proteins is applied at one location in an electric field, the negatively charged proteins will move towards the anode (+ pole) and the positively charged proteins will move towards the cathode (- pole). This makes separation of proteins easy. The distance that a particular protein migrates in an electric field depends on its molecular weight and charge, which in turn depends on the pH of the buffer solutions. The greater the charge and the less the molecular weight, the greater is the electrophoretic mobility. It is quite unlikely that two proteins will be present in a mixture having the same charge and size.
Supporting Medium
In electrophoresis various supporting media are used for the migration of charged particles. These include paper, starch, agar gel, cellulose acetate, polyacrylamide, etc. The supporting medium should be made wet with the appropriate buffer solution. One end of the supporting medium is connected to anode and the other end to cathode. The mixture to be separated is placed on the supporting medium.
Buffers
Buffer is a solution of a weak acid and one of its salts. Various buffers are used in electrophoresis. The function of a buffer is to keep the supporting medium wet so that a current will flow and maintain a pH during the experiment. This will help the substance to migrate under fairly constant conditions. The pH of the buffer must not be that of the isoelectrlc point of the substance (lsoelectric point is that pH at which the substance behaves electrically neutral). Though there are different types of electrophoresis, the one that is widely used in laboratories is called zone electrophoresis. In this type, the particles move through a solid supporting medium such as filter paper, cellulose acetate, agarose, starch, polyacrylamide, etc. Zone electrophoresis was developed by Tiselius in 1937. The main advantage of this electrophoresis is the complete physical separation of the components in a mixture. After electrophoresis, the spots, zones or bands of colourless materials can be located by treatment with some suitable colouring reagents. The electrophorogram can be photographed or preserved. Quantitative estimation of material in the zone or spot is possible by photometric scanning or by elution. Two common method of zone electrophoresis are
Paper Electrophoresis
In this method, a strip of filter paper moistened with buffer solution acts as the supporting medium. The paper is suspended between two reservoirs of buffer solution with one end dipping into each solution. Electrodes are placed in the buffer reservoirs, so that one reservoir functions as the anode and the other as the cathode. A small amount of mixture to be separated is applied near the centre of the paper and the voltage is applied from a power suppy unit. The buffer solution conducts the current and the components in the mixture move in one direction or the other at different rates determined by their physical properties.

Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gels are synthetic polymers formed from low molecularweight chemicals. It can be prepared in varying concentrations. The gels are prepared either as flattened slabs or as cylindrical columns. The molecules in the gel form a network and migrating macromolecules must squeeze through narrow tortuous passages. The pore size is regulated by varying the concentration of the gel. The rate of movement of molecules through the gel depends only on the molecular weight of the molecules. Smaller molecules pass through the gel more easily and migration rate increases as molecular weight decreases. PAGE yields very high resolution of proteins. it is a powerful tool for protein separation and characterization .