Sensitive techniques for the detection of genetic variation in human isozymes.
The principal means by which genetic variation of enzyme proteins has been revealed is the physicochemical separation of isozymes in an electrical field followed by their specific detection. This approach is widely applicable to all types of species and almost every conceivable kind of tissue homogenate or protein secretion. In the human organism alone the products of more than 200 enzyme loci have been investigated by this means and more than 40 genetically determined human isozyme polymorphisms have been identified. Much of this progress has been achieved by the use of conventional gel electrophoresis methods and isozyme detection schemes that depend on enzyme-catalyzed conversion of substrate into chromogens or other types of visible product. However, the functional isozyme detection systems are often of limited sensitivity and are dependent on the good condition of the biological material under analysis and the retention of adequate enzyme activity. Also in some cases, especially where the final product of the detection reaction is not an insoluble compound, the staining may be so diffuse as to offset the high resolving power of the most effective protein separation methods such as isoelectric focusing in ultranarrow pH gradients. The recent development of alternative protein detection methods based on immunoblotting offers a new general approach to isozyme analysis and overcomes some of the limitations of isozyme detection by functional staining methods. The immunoblot methodology is applicable to proteins separated electrophoretically on denaturing gels and on native gels, including proteins separated by isoelectric focusing, and the fidelity of the high-resolution separation techniques appears to be preserved for most proteins by passive or electroblot transfer to nitro-cellulose filters. Furthermore this approach provides considerable flexibility in the sensitivity and the nature of the final reporter signal that is employed. For example, the reporter signal can be amplified by the use of appropriate intermediate reagents to provide a level of detection sensitivity for isozyme proteins which should, even with the present procedures, be far in excess of that which is obtained even by the best functional isozyme staining system. Also the use of an evanescent reporter system such as chemiluminescence may allow multiple analyses of the same immunoblot and hence examination of several different isozyme systems by sequentially using different primary antibodies.
D005784:Gene Amplification; D005796:Genes; D014644:Genetic Variation; D006801:Humans; D007118:Immunoassay; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008722:Methods
D007527:Isoenzymes