title: Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix.
abstract: 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.
authors.affiliation: Institute of Microbiology and Genetics, Vienna Biocenter, A-1030 Vienna, Austria. vladimir@gem.univie.ac.at
authors.forename: Vladimir R
authors.initials: VR
authors.lastname: Kaberdin
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: McDowall
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005782:Gels; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D005782:Gels; D009838:Oligodeoxyribonucleotides; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D012259:Ribonuclease, Pancreatic; D000469:Alkaline Phosphatase