Detection and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native polyacrylamide gel electrophoresis
Detection and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native polyacrylamide gel electrophoresis
复制标题
DOI：
10.1002/elps.200500040
复制
发表时间：
2005-08-01
期刊：
影响因子：
2.9
通讯作者：
Appanna, V
中科院分区：
文献类型：
--
作者：
Bériault, R;Chénier, D;Appanna, V
我们描述了一个蓝色的本地聚丙烯酰胺凝胶电泳技术，可以方便地检测，定量和纯化的三个NADPH生产酶。同时检测葡萄糖6-磷酸脱氢酶、苹果酸酶和依赖NADP的异柠檬酸脱氢酶。活性染色的基础上形成的NADPH从各自的基板和随后沉淀的甲使酶活性的相对定量，而考马斯染色的一维或二维凝胶有助于监测与这些酶活性相关的蛋白质的量。该技术提供了一种简单有效的途径来获得用于进一步分析的均质蛋白，并且还使得能够在各种细胞系统中筛选这些NADPH产生酶。
We describe a blue native polyacrylamide gel electrophoretic technique that allows the facile detection, quantitation and purification of three NADPH-producing enzymes. Glucose 6-phosphate dehydrogenase, malic enzyme and NADP-dependent isocitrate dehydrogenase were detected simultaneously. Activity staining based on the formation of NADPH from the respective substrates and the subsequent precipitation of formazan enabled the relative quantitation of enzymatic activities, while Coomassie staining on one-dimensional or two-dimensional gels helped monitor the amount of protein associated with these enzymatic activities. This technique provides a simple and effective route to obtain homogeneous protein for further analyses and also enables the screening of these NADPH-producing enzymes in various cellular systems.