483 Lecture 7 - PAGE on GOT -- Expt 4 Part A
1. Native PAGE
PAGE is probably the most highly resolving electrophoretic method yet developed for separating proteins. In electrophoresis, proteins are separated by charge-density. In gel electrophoresis, the support media (the gel) also contributes to the separation power of the method. Using the organic monomer, acrylamide, to make the gel by free radical polymerization results in very uniform pore sizes which can be reproduced each time a gel is poured without a lot of variation.
How to make a PAGE Gel.
Chemistry:
Since the pores in a PAGE gel are the size of proteins, molecular sieving contributes to the resolving power of PAGE. Consequently, PAGE is a high resolution method and one of the best available for separating complex mixtures of proteins, while using simple equipment.
Models of PAGE Gel and Setups:
Native PAGE is used to determine the purity of a protein or enzyme.
This method of gel electrophoresis allows one to separate native proteins according to difference in their charge density. The buffer in the gel is suitable for maintaining the protein in its Native state. Thus, for enzymes their activity can be assayed after the electrophoretic separation. All proteins present in the gel can be visualized using a general protein stain. By comparison of identical gels stained specifically for the enzyme of interest and gels stained by the general protein stain, you can evaluate the purity of an enzyme preparation. The evaluation can be done in a quantitative manner by comparing relative mobilities of the enzyme-stained protein band and the protein stained protein bands. Relative mobility is defined as the distance moved by the protein band of interest as compared to the distance moved by the dye front (a low molecular weight dye which is highly charged is used to mark the electrophoretic front).
NOTE WELL:
- NATIVE PAGE is NOT a method for determining native molecular weight. Proteins do not separate according to molecular weight under the conditions of the native gel. Thus, native molecular weight of a protein can not be obtained by doing a single native PAGE gel.
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