SignaTECT® cdc2 Protein Kinase Assay System Technical Bulletin

Literature # TB227

The SignaTECT® cdc2 Protein Kinase Assay System uses a biotinylated peptide substrate derived from histone H1 to measure cdc2 kinase activity directly in cell lysates. This substrate (PKTPKKAKKL) is highly selective for cdc2 and possibly cdk2 and cdk3, providing the specificity necessary to perform cdc2 kinase assays on a variety of biological samples including crude cell lysates. The radiolabeled, phosphorylated substrate is recovered from the reaction mix with the SAM²® Biotin Capture Membrane (SAM²® Membrane), which is a novel streptavidin matrix. The SAM²® Membrane is prenumbered and partially cut so that individual squares can be easily identified, separated and placed in scintillation vials. Alternatively, the intact SAM²® Membrane can be analyzed using a phosphorimaging system or by conventional autoradiography. Olomoucine, a specific inhibitor of a subset of cdk enzymes including cdc2, cdk2 and cdk5, is provided in the system and can be used to confirm specificity of the reaction. With this assay, the analysis of cdc2 kinase is simple, rapid and amenable to high throughput. The assay detects olomoucine-sensitive cdc2 kinase activities at levels approaching pmol/min/µg of mitotic cell lysate protein.