Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix.

In the postgenomic era, the comprehensive proteomic analysis of metabolic and signaling pathways is inevitably faced with the challenge of large-scale identification and characterization of polypeptides with a particular enzymatic activity. Previous work has shown that a wide variety of enzymatic activities of microbial, plant, and animal origin can be assigned to individual polypeptides using in-gel activity staining (zymography). However, a number of limitations, such as special substrate requirements, the lack of a standard procedure, and difficulties in distinguishing enzymes with overlapping activities have precluded the widespread use of zymography as a routine laboratory method. Here we demonstrate that, by employing small-defined substrates that are covalently attached to the gel matrix, we can largely overcome the aforementioned problems and assay readily a number of different classes of enzymatic activities within gels after standard SDS-polyacrylamide electrophoresis. Moreover, this development is compatible with the two-dimensional separation of proteins and thus has great potential in the high-throughput screening and characterization of complex biological and clinical samples.