Define Polyacrylamide gel electrophoresis (PAGE)?
Acrylamide gel has the advantage over starch in that it is easier to prepare and is more inert, the pore size can be varied in a controlled manner and over a wider range, and the technique is similar with a shorter running time. In PAGE, gels are made by the free radical induced polymerization of the acrylamide monomer with N,N' methylenebisacrylamide as cross linking agent; in the presence of an initiator N, N, N1, N1- tetramethyl ethylenediamine (TEMED) in a buffer of selected pH and ionic strength and in the presence of a catalyst such as ammonium per sulphate to initiate polymerization as soon as it is introduced into the monomer. Photochemical catalysis with riboflavin and a bright light is an alternative. The rate of polymerization is proportional to the TEMED concentration. Electrophoresis can be carried out in cylindrical glass tubes or by using vertical or horizontal flat-bed equipment. In the simplest form of PAGE, a tube 3 to 10 cms long in which the gel has been polymerized is suspended vertically between an upper and a lower buffer reservoir. The buffer, which is the same in both reservoirs and the gel, has a pH (about 9 for protein) such that the macromolecules have net negative charges and hence migrate to the anode in the lower reservoir. Each sample which can contain as little as 10 µg of macromolecular material is dissolved in a minimal amount of a relatively dense glycerol or sucrose solution to prevent it from mixing with the buffer in the upper reservoir and is applied to the top of the gel.
A direct current of 300 V is passed through the gel till the components are separated into a series of discrete bands (30-90 minutes), the gel is removed from its holder, and the bands are visualized by an appropriate method. Using this technique a protein mixture of 0.1 to 0.2 mg can be resolved into as many as 20 discrete bands. The very large pores needed for the PAGE of large molecular mass compounds (> 200 kDa) requires gels with very low polyacrylamide concentrations (< 2.5%) and hence they are too soft to be usable. This difficulty is overcome by using agarose alone or mixed with polyacrylamide. For example, a 0.8% agarose gel is used for the electrophoretic separation of nucleic acids with molecular masses of upto 50,000 KDa. The bands resulting from a gel electrophoretic separation can be located by a variety of techniques.