Medical Books
The most widely used method for determining the purity of a protein is SDS-PAGE, polyacrylamide gel electrophoresis(PAGE) int the presence of the anionic detergent sodium dodecyl sulphate(SDS).
Electrophoresis separates charged bio molecules based on the rates at which they migrate in an applied electrical field.
For SDS-PAGE, acrylamide is polymerized and cross linked to form a porous matrix.
SDS denatures and binds to proteins at a ratio of one molecule of SDS per two peptide bonds. When used in conjunction with 2-mercaptoethanol or dithioreitol to reduce and break disulphide bonds, SDS seperates the component polypeptides of multimeric proteins.The large number of anionic SDS molecules, each bearing a charge of minus one, on each polypeptide overwhelms the charge contributions of the amino acid functional groups.Since the charge to mass ratio if each SDS polypeptide is approximately equal, the physical resistance each peptide encounters as it moves through the acrylamide matrix determines the rate of migration.Since larger complexes encounter greater resistance, polypeptides separate based on their relative molecular mass.Individual polypeptides trapped in the acrylamide gel are visualised by staining with dyes such as coomasie blue.