Abstract for SignaTECT^® cAMP-Dependent Protein Kinase (PKA) Assay System

Part# TB241
Printed in USA. Revised 6/09.
Instructions for Use of Product V7480.

The most common method to assay PKA activity involves measuring the transfer of ³²P-labeled phosphate to a protein or peptide substrate that is captured on phosphocellulose filters via weak electrostatic interactions. However, in the presence of multiple kinases (e.g., in a tissue extract), the ³²P-labeled peptides/proteins bound to the phosphocellulose filter may reflect kinase activity other than that of PKA. To increase specificity, the SignaTECT^® cAMP-Dependent Protein Kinase Assay (PKA) Assay System uses biotinylated Kemptide (LRRASLG), a peptide substrate derived from the in vivo substrate pyruvate kinase. This substrate is highly specific for PKA. The high affinity of Kemptide for PKA (K_m= 5–10μM) provides sufficient sensitivity to detect the enzyme at levels typically found within most biological samples. The ³²P-labeled, biotinylated substrate is recovered from the reaction mix with the SAM^2® Biotin Capture Membrane, which is a novel streptavidin matrix.