SDS gel electrophoresis and Western blotting.
Introduction
Electrophoresis is the migration of charged molecules in solution in response to an electrical field. (SDS PAGE) Polyacrylamide sodium duodecasulphate gel was used in this investigation in order to determine the molecular weights and possible identification using western blot of particular serum proteins. Using electrophoresis, provided a means of separating proteins of a given molecular weight (markers) and thus through this the molecular weight of the unknown serum proteins. The proteins were held within a porous gel which acts as a sieve by retarding the larger molecules whilst allowing migration freely through the pores of smaller molecules. The (PAGE) gels retain the larger molecules in the gel while the (SDS) denatures the proteins.
(SDS) is an anionic detergent which wraps around the polypeptide backbone and binds to the proteins and in so doing conferring a strong negative charge to the amino acid sequence in proportion to its length. (SDS) breaks up the two and three dimensional structure of the protein by adding a negative charge to all the amino acids straightened out. In denaturing, (SDS PAGE) separations, migration is determined not by intrinsic electrical charge of the polypeptide but by molecular weight between log molecular weight and (RF) distance migrated by the protein/ distance migrated by the dye front. It is usually necessary to reduce disulphide bonds in proteins before they adopt the random coil configuration necessary for separation by size; this is achieved with the addition of 2-mercaptoethanol or dithiothreitol which was carried out in this investigation. To be able to determine the subsequent separated serum proteins, Western blot is a technique that is used in order to make the proteins accessible to antibody detection
