In-gel phosphatase assay using non-denaturing two-dimensional electrophoresis.

We developed a method for detecting phosphatase activities in crude tissue extracts after separation of proteins by a novel non-denaturing two-dimensional electrophoresis. In the first dimension, protein samples were separated by a MicroRotofor, a liquid-phase isoelectric focusing, in the presence or absence of urea. In the second dimension, fractionated proteins by the MicroRotofor were resolved by a native polyacrylamide gel electrophoresis in the presence of 20 mM 2-mercaptoethanol. After electrophoresis, the polyacrylamide gel was directly immersed in a reaction mixture containing 4-methylumbelliferyl phosphate (MUP), a fluorogenic substrate, and phosphatase activities were detected as fluorescent bands. In this assay, a variety of phosphatase activities were clearly detected in gel when the tissue extracts were separated by the MicroRotofor in the presence of 1.5 M urea. Furthermore, after detecting phosphatase activities in polyacrylamide gel at neutral pH, its activities at acidic pH could be detected by immersing the gel in sodium citrate buffer (pH 3.0). Therefore, this method is a quite useful technique to analyze various phosphatases by sequential reactions with MUP under different conditions after sample separation by the two-dimensional electrophoresis.