A caution

Although IEF is a very valuable analytical and preparative technique, it can produce artifactual results. One problem is that proteins are very poorly soluble at or near their pi and so precipitation of components may occur during focusing. Also, apparently different isoforms of a protein (as revealed by focusing) can be due to conformational isomers, oxidation or reduction, or formation of complexes with other proteins or ampholytes. A good check for this is to apply the sample at different positions in the pH gradient and check for an identical pattern and to ensure IEF-isolated components retain their identity on repeated focusing.

See also: Antibodies, detection of; Monoclonal antibodies (mAbs); Protein separation techniques; SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

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