Phosphorylation assay is a widespread technique usually necessary for the identification of a specific kinase substrate and/or for the measurement of kinase activity. As an example of the technique, here we describe an assay aimed to test the phosphorylation of the myelin basic protein (MBP) by protein kinase C (PKC), which is overexpressed and purified fromNeurospora. The kinase is immunopurified fromNeurosporausing the expression vector pMYX2 and the FLAG epitope. The purified PKC and the MBP are then incubated in the presence of radioactive ATP, and the phosphorylated product is separated using the polyacrylamide gel electrophoresis technique.
