Introduction:
Electro- electricity, phoresis- to carry across. Electrophoresis is the migration of charged molecules in solution in response to an electric field. It is a method of separation of macromolecules like nucleic acids, carbohydrates, proteins, etc on the basis of charge and mass. It is a simple yet highly sensitive tol for separating & analyzing properties of molecules.
History:
1955 Introduction of starch gels
1959 Introduction of acrylamide gels (Raymond and winstraub)
1969 Introduction of SDS (Beer & John)
Late 1970s Agarose gels
1983 Pulse Field gel electrophoresis
Principle:
Separation of the large molecules depends on two forces charge and mass. When a biological sample, such as proteins/ DNA, is mixed in a buffer solution & applied to gel, these two forces act together. The electric current from one electrode repels the molecules while the other electrode simultaneously attracts the molecule. The frictional force of the medium (gel) acts as a “molecular sieve” separating the molecules by size. A gel is a colloid which serves as a solid support for migration. During electrophoresis, macromolecules are forced to move through the pores, when the electrical current is applied. Their rate of migration depends upon the strength of electric field, size and shape of molecules and relative hydrophobicity of the sample.
Instrumentation:
Two pieces of equipment are necessary for electrophoresis.
a) D.C. supply: It has different voltages 50V, 100V, switches & cathode & anode connecting points where in the electrophoretic unit could be connected via red and black wires.
b) Buffer reservoir system: System consists of upper and lower buffer reservoirs with provision for suspending the gel. The only electrical connection between reservoirs is via the gel. Platinum electrodes are positioned in each reservoir. Most buffers maintain pH 8-9.
Types of gel electrophoresis:
a) Agarose gel electrophoresis: Agarose gel consists of purified large MW polysaccharide (from agar). It is a large pore gel and which is why it is frequently used for large DNA molecules.
b) Starch gel electrophoresis: They are prepared by heating and cooling a mixture of partially hydrolysed starch in an appropriate buffer. This causes the branched chains of the amylopectin components of starch to interwine & form a semi-rigid gel
c) Polyacrylamide gel: Acrylamide gel first form linear chains and are then coplymerised with cross linker N’,N’-Methylene bisacrylamide in the presence of APS (Ammonium persulfate) and TEMED as catalyst.
