Phos-tag Affinity Electrophoresis for Protein Kinase Profiling
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Protein kinase profiling can provide a basis for understanding the molecular origins of diseases and, potentially, for developing
            tools for therapeutic intervention. It is therefore very important to develop advanced experimental procedures for convenient
            and accurate determination of the phosphorylation status of certain substrate proteins in the life sciences. Here, we introduce
            a method for protein kinase profiling by using a novel type of phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel
            electrophoresis (SDS-PAGE). The phosphate-affinity site is a polyacrylamide-bound dinuclear metal complex of a phosphate-binding
            tag molecule known as Phos-tag. The Phos-tag SDS-PAGE method permits detection of changes in the mobility of phosphorylated
            proteins in comparison with their nonphosphorylated counterparts and thereby allows quantitative analysis of protein kinase
            reactions without any special apparatus, radioactive isotopes, or chemical labels. If a kinase reaction occurs at one residue
            of a substrate protein, the monophosphorylated and nonphosphorylated forms can be simultaneously detected as two migration
            bands on a Phos-tag SDS-PAGE gel. In the case of hyperphosphorylation, the phosphorylated products appear as multiple migration
            bands, depending on the phosphorylation status in terms of the numbers and the positions of attached phosphate groups. This
            article discusses applications of label-free kinase activity profiling by the Phos-tag SDS-PAGE method in the analysis of
            phosphorylated substrates derived from various kinase reactions. The resolving power of the affinity electrophoresis provides
            detailed information that leads to an overview of the kinase-dependent dynamics of various substrate proteins.
         
      










