Activity Gels: Reformation of Functional Proteins in SDS-Polyacrylamide Gels
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is an important technique for analyzing the complexity of polypeptides in crude mixtures and for determining the subunit structures of purified proteins (1). The two-dimensional gel system developed by O’Farrell, that combines isoelectric focusing in urea with SDS-PAGE, offers the additional advantage of greater resolution than SDS-PAGE alone (2,3). Even small amounts of proteins can be resolved as discrete spots, and microheterogeneity in related polypeptides can be easily detected. However, the power of these techniques is limited by the fact that the proteins remain denatured in the gel and only abundant proteins can be identified by their positions on the gels. One recent approach to the detection of nonabundant proteins in a crude mixture, Western blotting, is the use of antibodies to identify specific antigens after separation on SDS-gels and transfer to nitrocellulose paper (4). This technique, though powerful, has the limitation of requiring antibodies, which usually necessitates prior purification of a specific protein of interest. An alternative approach is to detect isoproteins in a gel by their biochemical activities. Our laboratory first developed these procedures for identification of enzymes in high-resolution denaturing gels and recently they have been employed for other enzymes.
KeywordsCrude Extract Mixed Micelle Dodecyl Alcohol Adenylate Kinase Molecular Heterogeneity
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