Enzo Life Sciences
PKA kinase activity kit
ADI-EKS-390A
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Protein Kinase A is a Ser/Thr protein kinase that phosphorylates multiple substrates in the cell. This kinase is activated by cAMP. The kinase exits as a tetrameric protein consisting of two catalytic and two regulatory subunits. cAMP generated by adenylate cyclases binds to the regulatory subunit and this leads to the dissociation of the regulatory subunits from the catalytic subunits. The dissociated catalytic subunits are then active to phosphorylate downstream targets. In the present experiment, the phosphorylation of a synthetic peptide by purified PKA was measured by a colorimetric assay. Since this assay is a non-radioactive assay, it is much easier to perform multiple measurements of endogenous PKA activity after certain manipulations without the tiring radioactive assays.
Colorimetric assay
Purified kinase
This experiment was done as a pilot experiment to test the specificity of the kit. The phosphorylation assay was performed either in the absence or presence of purified PKA according to the manufacturer's instructions.
Follow the manufacturer's instructions
A specific colorimetric signal (indicates the phosphorylation of a synthetic PKA substrate) was observed only in the presence of purified PKA.
None
High signal:background noise
The protocol involves multiple steps but performs well with purified PKA and and moderately with crude extracts.
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