UNIT 13.12 In-Gel Phosphatase Assay Using Fluorogenic and Radioactive Substrates
Published Online: 1 AUG 2011
Copyright © 2011 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Kameshita, I. 2011. In-Gel Phosphatase Assay Using Fluorogenic and Radioactive Substrates. Current Protocols in Protein Science. 65:13.12:13.12.1–13.12.10.
- Published Online: 1 AUG 2011
- Published Print: AUG 2011
To investigate the regulatory mechanisms of cellular signaling by protein phosphorylation, it is important to analyze protein phosphatases, as well as protein kinases expressed in cells and tissues. In this unit, two different types of in-gel phosphatase assays are described. The first is an in-gel phosphatase assay using fluorogenic substrates. Protein samples containing phosphatase activities are resolved by native polyacrylamide gel electrophoresis (native-PAGE) and phosphatase activities detected in situ using fluorogenic substrates, such as 4-methylumbelliferyl phosphate (MUP) or 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). The other assay is an in-gel phosphatase assay using 32P-labeled substrates. In this method, protein samples are resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using polyacrylamide gels containing 32P -labeled substrates, renatured in situ, and the dephosphorylating activities detected by autoradiography. Each method has advantages and disadvantages that are discussed in the commentary. Curr. Protoc. Protein Sci. 65:13.12.1-13.12.10. © 2011 by John Wiley & Sons, Inc.
- in-gel assay;
- polyacrylamide gel electrophoresis