Question

. Most of the protein purification methods described in this lecture are used to purify enzymes in their. native state. Why would the use of SDS-polyacrylamide gel electrophoresis be unlikely to lead to the successful purification of an active enzyme?

Answer 1

ANSWER:-In SDS-PAGE, proteins are drawn through the gel by an electric current. In the process, however, all proteins become denatured. In this case, we would not be able to purify an active enzyme. Some crystalline forms of enzymes are catalytically active and thus are able to carry out the same chemical reactions as they can in solution.

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. Although covalent structural features of resolved proteins can be determined with SDS-PAGE, functional properties are destroyed, including the presence of non-covalently bound metal ions. To address this shortcoming, blue-native (BN)-PAGE has been introduced. This method retains functional properties but at the cost of protein resolving power. To address the need for a high resolution PAGE method that results in the separation of native proteins, experiments tested the impact of changing the conditions of SDS-PAGE on the quality of protein separation and retention of functional properties.