Longley M J, Mosbaugh D W
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Methods Enzymol. 1993;218:587-609. doi: 10.1016/0076-6879(93)18043-c.
We have presented several protocols for producing an in situ activity gel that allows detection of various DNA-metabolizing enzymes. Both nondenaturing polyacrylamide and SDS-polyacrylamide activity gel electrophoresis procedures were detailed. Combining the use of defined [32P]DNA substrates with product analysis, these procedures detected a wide spectrum of enzymatic activities. The ability to detect 7 different catalytic activities of 15 different enzymes provides encouragement for expanded applications. It is hoped that others will find this technique applicable for detecting these enzymes and other activities in different biological systems. The modification of DNA in situ and the creation of intermediate substrates within activity gels should prove extremely useful for dissecting the enzymatic steps of DNA replication, repair, recombination, and restriction, as well as the metabolic pathways of other nucleic acids.
我们已经介绍了几种用于制备原位活性凝胶的方案，该凝胶可用于检测各种DNA代谢酶。详细阐述了非变性聚丙烯酰胺和SDS-聚丙烯酰胺活性凝胶电泳程序。将特定的[32P]DNA底物与产物分析相结合，这些程序检测到了广泛的酶活性。能够检测15种不同酶的7种不同催化活性，为扩大应用提供了鼓舞。希望其他人会发现这项技术适用于检测不同生物系统中的这些酶和其他活性。原位DNA修饰以及活性凝胶中中间底物的产生，对于剖析DNA复制、修复、重组和限制的酶促步骤以及其他核酸的代谢途径应该会非常有用。
