用于检测和纯化吡啶核苷酸连接脱氢酶的天然凝胶活性染色及制备电泳方法。

Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases.

作者信息

Seymour J L, Lazarus R A

机构信息

Biomolecular Chemistry Department, Genentech, Inc., South San Francisco, California 94080.

出版信息

Anal Biochem. 1989 May 1;178(2):243-7. doi: 10.1016/0003-2697(89)90632-5.

DOI:10.1016/0003-2697(89)90632-5

PMID:2473665

Abstract

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.

摘要

本文描述了一种用于检测聚丙烯酰胺凝胶中吡啶核苷酸连接脱氢酶的活性染色方法。将凝胶与底物和辅因子孵育后，在紫外光下观察条带，还原型辅因子发出荧光，氧化型辅因子呈黑色。所述方法适用于任何与NAD或NADP连接的脱氢酶；酶可以在氧化或还原方向上进行测定。还描述了一种使用活性染色的制备型聚丙烯酰胺凝胶系统，该系统可用作脱氢酶的通用纯化方法。制备型凝胶用双丙烯酰半胱胺交联。在找到并切除感兴趣的条带后，通过添加硫醇可以断开这些交联。然后，通过吸附到合适的树脂上，将感兴趣的蛋白质与溶解的丙烯酰胺分离。