In-gel phosphatase assay using fluorogenic and radioactive substrates.

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 (32)P-labeled substrates. In this method, protein samples are resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using polyacrylamide gels containing (32)P -labeled substrates, renatured in situ, and the dephosphorylating activities detected by autoradiography. Each method has advantages and disadvantages that are discussed in the commentary.