SignaTECT® cAMP-Dependent Protein Kinase (PKA) Assay System Technical Bulletin
Literature # TB241
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 or 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 (Km= 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 SAM2® Biotin Capture Membrane, which is a novel streptavidin matrix.
Printed in USA. Revised 1/13.