Native PAGE : Polyacrylamide gel electrophoresis II Protein Electrophoresis

While SDS–PAGE is the most frequently used gel system for studying proteins, the method is of no use if one is aiming to detect a particular protein (often an enzyme) on the basis of its biological activity, because the protein (enzyme) is denatured by the
SDS–PAGE procedure. In this case it is necessary to use non-denaturing conditions. In native or buffer gels, polyacrylamide gels are again used (normally a 7.5% gel) but the SDS is absent and the proteins are not denatured prior to loading. Since all the
proteins in the sample being analysed carry their native charge at the pH of the gel
(normally pH 8.7), proteins separate according to their different electrophoretic mobilities and the sieving effects of the gel. It is therefore not possible to predict the behaviour of a given protein in a buffer gel but, because of the range of different charges and sizes of proteins in a given protein mixture, good resolution is achieved.
The enzyme of interest can be identified by incubating the gel in an appropriate
substrate solution such that a coloured product is produced at the site of the enzyme.
An alternative method for enzyme detection is to include the substrate in an agarose gel that is poured over the acrylamide gel and allowed to set. Diffusion and interaction of enzyme and substrate between the two gels results in colour formation at the site of
the enzyme. Often, duplicate samples will be run on a gel, the gel cut in half and one half stained for activity, the other for total protein. In this way the total protein content of the sample can be analysed and the particular band corresponding to the
enzyme identified by reference to the activity stain gel.

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