Here we describe a convenient, inexpensive, and non-hazardous method for the measurement of the kinase activity of the catalytic subunit of cAMP-dependent protein kinase (PKACα). The assay is based on the separation of a substrate peptide labeled with a strong chromophore from the phosphorylated product peptide by high-performance liquid chromatograph (HPLC) and quantification of the product ratiometrically at a wavelength in the visual spectrum (Vis). The utility and reliability of the HPLC-Vis assay were demonstrated by characterizing the kinetic parameters (K, V) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC, K) of the known PKACα inhibitors H89 and PKI(5-24). The advantages of this assay are that it is convenient and inexpensive, uses readily synthesized or commercially available substrates that are shelf-stable, uses a common piece of laboratory equipment, and does not require any hazardous materials such as radioactive γ-P-ATP. The assay format is also highly flexible and could be adapted for the testing of many different kinases by changing the peptide substrate sequence.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889107 | PMC |
| http://dx.doi.org/10.1016/j.ab.2017.06.001 | DOI Listing |
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