1. General principle: Many important biological molecules such as amino acids, peptides, proteins, nucleotides and nucleic acids posses ionisable groups and can therefore be made to exists in solution as electrically charged species either as cations (+) or anions (-). When a mixture of these components are subjected to electric field, they migrate differentially and thus can be separated.
(i) low voltage thin sheet electrophoresis; (ii) high voltage electrophoresis, (iii) gel electrophoresis – native poly acrylamide gel electrophoresis and sodium dodecyl sulphate (SDS) poly acrylamide gel electrophoresis, (iv) isoelectric focussing and (v) isotachophoresis.
The most commonly used electrophoresis is gel electrophoresis. In this technique either agarose or poly acrylamide is used as supporting media. 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. Gel dimensions are mostly 12 cm x 14 cm with a thickness of 0.5 mm to 1.0 mm. A plastic comb is placed in the gel solution and removed after polymersization to provide loading wells for the samples. When the apparatus is assembled, the lower electrophoresis tank buffer surrounds the gel plates and effect cooling of the gel plates.
In horizontal gel system, the gel is cast on a glass or a plastic sheet and placed on a cooling plate. Connection between the gel and electrode buffer is made by using a thick wetted filter paper (wick). The power pack supplies direct current between the electrodes in the electrophoresis unit. All electrophoresis is carried out in appropriate buffer to maintain constant state of ionization of the components being separated. Any variation in pH may alter the over all charge and so the mobility of the molecules being separated (Fig. 10.7).
Agarose Gels: Agarose is a linear polysaccharide made up of repeating units of agarobiose which contains galactose and 3, 6 anhydro galactose. This is isolated from seaweeds. Agaroses gel is usually prepared at the concentration of 1-3% solutions. The gels can be prepared by suspending dry agarose in suitable 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. Agarose gels are used for the electrophoresis of both proteins and nucleic acids.
Polyacrylamide Gels: Electrophoresis in acrylamide gels is referred as PAGE being an abbreviation for PolyAcrylamide Gel Electrophoresis. Polyacrylamide gels are prepared by dissolving required quantity of acrylamide with a small amount of N, N’-methylene bisacrylamide in suitable buffer. The polymerization is initiated by ammonium persulphate and N, N, N’, N’-tetramethylene diamine (TEMED).The polymerization is free radical mediated reaction. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis is the most widely used method for analyzing protein mixture qualitatively. It is particularly useful for monitoring protein purification. SDS is an anionic detergent. Samples to be run on SDS-PAGE are first boiled for 5 minutes in sample buffer containing beta mercapitoethanol and SDS. The mercapto ethanol reduces any disulphide bridges and cleave the protein into different sub-units. So, by this electrophoresis different units of proteins can be identified.
· Proteins can be detected by using the dye-solution Coomassie Brilliant Blue R-250 (CBB). Staining is usually carried out using 0.1% CBB in methanol : acetic acid : water in the ratio 5:1:5. The protein bands will look blue in colour.
· Glycoproteins are detected by using periodic acid – schiff (PAS) stain. The bands will appear in red colour.
· Nucleic acids can be detected by using the fluorescent dye ethidium bromide. The nucleic acids bands will appear colourless in black background.