PKA (Protein Kinase A) Colorimetric Activity Kit, 96 Tests - FAQs

How does the PKA (Protein Kinase A) Colorimetric Activity Kit (Cat. No. EIAPKA) work?

The PKA (Protein Kinase A) Colorimetric Activity Kit (Cat. No. EIAPKA) is a solid-phase activity assay designed to measure PKA activity in cell lysates, tissue extracts, and buffered solutions. A recombinant PKA standard is provided to generate a standard curve for the assay, and all samples should be read off the standard curve. The kit uses an immobilized PKA substrate bound to a 96-well microtiter plate. Samples containing PKA will, in the presence of the supplied ATP, phosphorylate the immobilized PKA substrate. After a 90-min incubation followed by a wash, a rabbit antibody specific for the phospho-PKA substrate binds to the modified immobilized substrate. An antibody specific for rabbit IgG labeled with peroxidase is also added to the plate to bind to the rabbit anti-phospho-PKA substrate. After a short incubation and wash, substrate is added, and the intensity of the color developed is directly proportional to the amount of PKA in the samples and standards. A 96-well microplate reader capable of reading optical density at 450 and 650 nm is required for use of this kit.

The assay was characterized with human PKA, but can be used to measure PKA activity in samples from other species.

Note: PKA is a heterotetrameric cAMP-dependent protein kinase, made up of two regulatory subunits and two catalytic subunits in its inactive form. In its active state, the complex dissociates to into a dimeric regulatory subunit, and two enzymatically active catalytic subunits.

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