Electrophoretic techniques

Electrophoretic techniques. Advance Techniques in Biochemistry BCHM 431.

Introduction. The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions. Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge. The equipment required for electrophoresis consists basically of two items, a power pack and an electrophoresis unit..

Introduction--contd. Electrophoresis units are available for running either vertical or horizontal gel systems. Vertical slab gel units are commercially available and routinely used to separate proteins in acrylamide gels. The gel is formed between two glass plates that are clamped together but held apart by plastic spacers. The most commonly used units are the so-called minigel apparatus..

Introduction--contd. A plastic comb is placed in the gel solution and is removed after polymerisation to provide loading wells for up to 10 samples. When the apparatus is assembled, the lower electrophoresis tank buffer surrounds the gel plates and affords some cooling of the gel plates. The gel is cast on a glass or plastic sheet and placed on a cooling plate (an insulated surface through which cooling water is passed to conduct away generated heat). The power pack supplies a direct current between the electrodes in the electrophoresis unit..

Introduction--contd. All electrophoresis is carried out in an appropriate buffer, which is essential to maintain a constant state of ionisation of the molecules being separated. Any variation in pH would alter the overall charge and hence the mobilities of the molecules being separated. All electrophoresis is carried out in an appropriate buffer, which is essential to maintain a constant state of ionisation of the molecules being separated. Any variation in pH would alter the overall charge and hence the mobilities of the molecules being separated..

A typical horizontal electrophoretic apparatus. Cover Electrode - ve Compartment of buffer reservoir Gel Wick Electrode Cooling plate Fig. 10.2 A typical horizontal apparatus, such as that used for immunoelectrophoresis, isoelectric focussing and the electrophoresis of DNA and RNA in agarose gels..

SUPPORT MEDIA. The introduction of the use of gels as a support medium led to a rapid improvement in methods for analysing macromolecules. The earliest gel system to be used was the starch gel and, although this still has some uses, the vast majority of electrophoretic techniques used nowadays involve either agarose gels or polyacrylamide gels..

Agarose gels. Agarose is a linear polysaccharide made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is one of the components of agar that is a mixture of polysaccharides isolated from certain seaweeds. Agarose is usually used at concentrations of between 1% and 3%. Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel..

HO o OH o o OH o o HO OH ß-D- 3,6-anhydro- L-galactose galactose Agarose.