In-gel protein phosphatase assay using fluorogenic substrates

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URI http://shark.lib.kagawa-u.ac.jp/kuir/metadata/4979
Title
In-gel protein phosphatase assay using fluorogenic substrates
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Description

We developed a method for 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 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, denaturation/renaturation process of resolved proteins was necessary for detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphases 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.

Author
著者 Kameshita Isamu
著者(ヨミ) カメシタ イサム
著者(別表記) 亀下 勇
著者 Baba Hiromi
著者(ヨミ) ババ ヒロミ
著者(別表記) 馬場 裕美
著者 Umeda Yoshinori
著者(ヨミ) ウメダ ヨシノリ
著者(別表記) 梅田 嘉徳
著者 Sueyoshi Noriyuki
著者(ヨミ) スエヨシ ノリユキ
著者(別表記) 末吉 紀行
Publication Title
Analytical biochemistry
Volume
400
Issue
1
Start Page
118
End Page
122
Publisher
Elsevier
Published Date
20100501
ISSN
0003-2697
NCID
AA00524867
DOI
10.1016/j.ab.2009.12.035
Resource Type
Journal Article
Language
eng
Rights
Copyright c 2010 Elsevier Ltd
Text Version
author
Set
香川大学
Copyright (C) 2009 Kagawa University All rights reserved.