SDS not only provides proteins with a strong negative charge, but it also denatures the protein, thus giving each protein a roughly globular shape differences in protein shape that would affect electrophoretic mobility are eliminated. The effect before electrophoresis is to block protein interactions with other proteins, polymers such as nucleic acids and lipid, to dissociate multimeric proteins and to alter folding in protein monomers. Its action is to denature the protein by solubilizing it and effectively "coating" it with a negative charge. Sodium dodecyl sulphate (SDS), is an anionic detergent that binds to proteins.
Experimentally, we can eliminate the influence of charge on protein migration by providing all proteins with extreme negative charges. For example, globular proteins are retained less than rod-like proteins of the same molecular weight.
However, proteins which have a similar net charge separate nicely according to their size, provided they are of similar shape. In native gel electrophoresis, both charge and size determine the migration pattern in this technique excellent separation can be achieved, but unambiguous information about the protein size cannot be obtained. On the other hand, the net charge of a protein depends on the pH. Smaller proteins are retained less, and thus move faster. If proteins are separated through a gel matrix with varying pore size, migration depends on the size and shape of the protein. Size and charge of a protein determine its electrophoretic mobility. In protein electrophoresis, these factors tend to balance out. Larger particles move more slowly, and highly charged particles move more quickly. Rate of particle movement is proportional to the charge:mass ratio of the particle and to its frictional resistance. SDS-Polyacrylamide gel electrophoresisĮlectrophoresis is the process in which charged particles migrate through a solid or liquid matrix in response to application of an electric field. SDS polyacrylamide gel electrophoresis (SDS PAGE) will be used to assess the purification process and to determine the apparent molecular weights of the three apoproteins. Lipoprotein Analysis Week 2: Electrophoresis
0 kommentar(er)
