Biotechnology and Bioinformatics (1+1)

SDS – Poly acrylamide gel electrophoresis

Electrophoresis is a technique used for the separation of proteins based on the migration of charged proteins in an electric field. This technique is used as an analytical method. In this technique proteins can be visualized and also separated. The number of different proteins in a mixture or the degree of purity of a particular protein preparation can be estimated quickly. Also electrophoresis allows determination of crucial properties of a protein such as its iso-electro point and approximate molecular weight.

Electrophoresis of proteins is generally carried out in gels made up of the cross linked polymer polyacrylamide. The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In electrophoresis, the force moving the macromolecule is the electrical potential. The electrophoresis mobility of the molecule, μ is the ratio of the particle, V, to the electrical potential (E).

μ = V/E

The migration of a protein in a gel during electrophoresis is therefore a function of its shape and its size. Normally SDS (Sodium dodecyl sulphate), a detergent is used for the estimation of purity and molecular weight. SDS binds to most proteins (probably by electrophoretic interactions) in amounts roughly proportional to the molecular weight of the protein i.e. 1 molecule of SDS for every two amino acid residues. The bound SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificantly. The native conformation of a protein is altered when SDS is bound and most proteins assume a similar shape. Electrophoresis in the presence of SDS therefore separate proteins almost exclusively on the basis of mass (molecular weight) with smaller polypeptides migration more rapidly. After electrophoresis the proteins are visualized by adding a dye such as coomassie blue, which binds to proteins, but not to the gel itself. By comparing with the position of the known protein, the molecular weight of the unidentified protein can be measured.

Polyacrylamide gels are formed by the linking co-monomer bisacrylamide. The gel consists of lower ‘separating gel’ and upper ‘stacking gel’. The pH of the buffers is adjusted such that the protein sample is concentrated in the stacking gel(pH 6.8) and starts moving to the separating gel (pH 8.8). The proteins unstuck and begin to separate according to their molecular weights. Treatment of proteins with the detergent SDS (Sodium dodecyl sulphate) and a reducing agent (μ-mercaptoethanol or ditiothreitol) changes their three dimensional shape into simple rod-like structures.