In-gel protein phosphatase assay using fluorogenic substrates
In-gel protein phosphatase assay using fluorogenic substrates
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DOI：
10.1016/j.ab.2009.12.035
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发表时间：
2010-05-01
影响因子：
2.9
通讯作者：
Sueyoshi, Noriyuki
中科院分区：
文献类型：
--
作者：
Kameshita, Isamu;Baba, Hiromi;Sueyoshi, Noriyuki
我们开发了一种在聚丙烯酰胺凝胶电泳后使用荧光底物检测磷酸酶活性的方法。当磷酸酶如Ca 2 +/钙调蛋白依赖性蛋白激酶磷酸酶（CaMKP），蛋白磷酸酶2C（PP 2C），蛋白磷酸酶5（PP 5），和碱性磷酸酶在SDS的情况下，通过聚丙烯酰胺凝胶电泳和凝胶孵育的荧光底物，如4-甲基伞形酮磷酸（MUP），所有这些磷酸酶的活性可以在原位检测。尽管6，8-二氟-4-甲基伞形酮磷酸酯（DiFMUP）以及MUP可用作凝胶内测定的荧光底物。MUP显示较低的背景荧光。使用该程序，在各种粗样品的电泳后观察到对应于内源性磷酸酶的几个荧光带。凝胶内磷酸酶测定也可用于检测SDS-聚丙烯酰胺凝胶电泳分离的蛋白磷酸酶。然而，在这种情况下，解析蛋白质的变性/复性过程对于磷酸酶活性的检测是必要的。该方法可用于检测细胞提取物中表达的CaMKP等可复性蛋白磷酸酶。本发明的荧光凝胶内磷酸酶测定法是非常有用的，因为不需要放射性化合物或特殊仪器。(C)2009 Elsevier Inc. All rights reserved.
We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay. MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required. (C) 2009 Elsevier Inc. All rights reserved.