In-gel protein phosphatase assay using fluorogenic substrates.
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.
Kameshita
Isamu
I
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan. kamesita@ag.kagawa-u.ac.jp
Baba
Hiromi
H
Umeda
Yoshinori
Y
Sueyoshi
Noriyuki
N
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005782:Gels / Q000737:chemistry*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D000071636:Protein Phosphatase 2C; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
C119285:6,8-difluoro-4-methylumbelliferyl phosphate; D005456:Fluorescent Dyes; D005782:Gels; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C005359:4-methylumbelliferyl phosphate; D006923:Hymecromone; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; C089641:protein phosphatase 5