ELECTROPHORESIS

ELECTROPHORESIS

Electrophoresis  is the analytical technique applied for the separation of charged molecules, based on their movement in an electric field ; positively charged particles move to the cathode, and negatively charged ones to the anode. This makes their separation easy. It is a very convenient technique for analysing and purifying several kinds of biomolecules, especially peptides, proteins, nucleotides and nucleic acids. Separation of the constituents of mixtures or solutions occurs at pH values .above or below the isoelectric point.

Electrophoresis relies on the principle that biological molecules in solu­tion carry a net electric charge (except at the isoelectric point). If two elec­trodes are placed in such a solution, and an electric field is applied, the charged particles move towards oppositely charged poles at different rates and get localized in different zones. Their movement is greatly influenced by their shape, size, molecular weight and electric charge, and also by the matrix of the gel support and the applied electric field. The greater the charge and the lesser the molecular weight, the greater would the electrophoretic mobility of the particles. The charge, in turn, depends on the pH of the buffer solution. A molecule with a double charge will move at twice the speed of a molecule with a single charge. This difference is the basis for the separation of the differ­ent molecules of mixtures or solutions. It is very unlikely that two different kinds of molecules of a mixture will have exactly the same size, molecular weight and charge level.

Modern elec­trophoretic techniques use wetted filter paper or a polymerized gel - like matrix (acrylamide gel, starch gel, cellulose acetate gel, agarose gel, silica gel, etc.) as an inert support medium. The sample to be analysed is applied to this medium as a spot or as a thin band.

The movement of charged molecules in an applied electric field is repre­sented by the equation

Thus , charged particles move at a velocity which depends directly on the applied electric field or voltage (E) and charge (q) , but inversely on a counteracting force generated by the viscous drag (f)

Types of electrophoresis

All electrophoretic methods are based on the same principle. So, they differ from each other only in the nature of the support medium. Some common types of electrophoretic methods are paper electrophoresis, polyacrylamide gel electrophoresis (PAGE - significant in the separation of pro­teins ), agarose gel electrophoresis (AGE - important in the separation of nucleic acids), sodium dodecyl sulphate - polyacrylamide gel electrophore­sis (SDS- PAGE), significant in the measurement of the molecular weight of biomolecules), pulsed field gel electrophoresis (PFGE), significant in the sepa­ration of large chromosomal DNA, capillary electrophoresis (CE- combina­tion of the resolving power of electrophoresis with the speed of high perfor­mance liquid chromatography to analyse very small samples ), etc.

Paper electrophoresis

This is the electrophoretic  method in which a filter paper wetted with a buffer solution forms the support medium. The pa­per spans between two reservoirs of the buffer solu­tion, with its ends immersed in the solution. A small amount of the solu­tion or mixture to be analysed is placed somewhere near the centre of the paper. Now, electrodes are placed in the reser­voirs of the buffer and a voltage is applied. The charged constituents of the mixture or solution move to their respective poles at different velocity. This brings about their separation.

 Polyacrylamide gel electrophoresis (PAGE)

PAGE is the electrophoretic method in which polyacrylamide gel is used as the support medium. Polyacrylamide gel is a synthetic polymer (prepared by the free radical polymerisation of acrylamide and the cross linking agent methylene-bis-acrylamide). Polyacrylamide gels are produced either as col­umns or as slabs. Slab gels are now more widely used than column gels. A slab gel is more convenient, because several samples can be analysed on it; only a single sample can be analysed on a tube (column) gel.

Acrylamide gel is something like a spongy network. Macromolecules can­not freely pass though it, but can only squeeze through its narrow channels. The rate of this movement depends largely on the size and molecular weight of the migrating molecules. Smaller the molecules and lower their molecular weight, faster would be their movement.

In PAGE gel column or gel slab is inserted vertically between two buffer reservoirs. The upper reservoir usually contains the anode, and the lower one contains the cathode. The sample to be analysed is placed on the gel slab or gel tube and a voltage is applied. The charged molecules of the sample move to their respective poles.PAGE is significant in that in it separation of the sample components involves both molecular sieving and electrophoretic movement So, it results  in enhanced resolution of sample components The order of molecular movement in gel filtration and PAGE is different. In gel filtration large mol­ecules move through the matrix faster than small molecules, but the reverse is the order in PAGE. Usually, PAGE is used for the separation of large mol­ecules, such as proteins.