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 is again used as a support matrix (normally at 7.5%), 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 sub strate 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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مواضيع ذات صلة

Androgen Biosynthesis

Androgens and Related Molecules

Two-Dimensional Polyacrylamide Gel Electrophoresis

Isoelectric Focussing Gels

Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis

Transition Metals are Essential for Health

Individual Regulatory Events Combine to Form Sophisticated Control Networks

Regulation of Enzyme Quantity

Biochemical Evaluation of Cobalamin and Folate Deficiencies

Compartmentation Ensures Metabolic Efficiency & Simplifies Regulation

Regulation of Metabolite Flow can be Active or Passive

Catabolism of Catecholamines