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- Assay Type Activity Assay
- Sample Types Cell Lysates, Tissue Extracts, Purified Systems
- Sensitivity 0.037 Units/mL
- Species Identical across all species
- Assay Duration 3 Hours
- Samples/Plate 42 in Duplicate
- Readout Colorimetric, 450 nm
- Standard Curve
The Protein Kinase A (PKA) Colorimetric Activity Kit quantitatively measures PKA activity in cell lysates, tissue extracts, and purified systems. The Protein Kinase A (PKA) Colorimetric Activity Kit is an Activity Assay with a run time of 3 hours. Please read the complete kit insert for more information before performing this assay.
Use our provided PKA Standard to generate a standard curve for the assay. Pipette the standards or diluted samples in the kinase assay buffer into a transparent microtiter plate with bound PKA substrate. Add the reconstituted ATP to each well, tapping the plate to ensure sufficient mixing of reagents. Then incubate the sealed plate at 30°C shaking for 90 minutes. After the 90-minute incubation, add the Donkey anti-Rabbit IgG HRP Conjugate and the Rabbit Phospho PKA Substrate Antibody to each well and incubate the plate at room temperature for 60 minutes.
After the 1-hour incubation, wash away the excess phospho-PKA-peroxidase conjugate and add the TMB substrate. The TMB substrate reacts with the bound phospho-PKA-peroxidase conjugate generating a signal detected by a plate reader at 450nm. Use the intensity and the standard curve to calculate the PKA activity in the samples.
The expressed PKA holoenzyme comprises two catalytic (C) and two regulatory (R) subunits and is activated when cAMP levels rise following stimulation of G Protein-coupled receptors and adenylyl cyclase. The subcellular localization of the enzyme regulates the phosphorylation of specific substrates by the C subunit of activated PKA through the binding to the scaffolding A kinase-anchoring proteins (AKAPs). In its inactive state, the pseudosubstrate sequences on the R subunits stop the activity of the C subunits. The active monomeric C subunits are released upon cAMP binding to the R subunits. PKA shares substrate specificity with Akt (PKB) and PKC. Substrates that PKA phosphorylates include Bad (Ser155), CREB (Ser133), and GSK-3 (GSK-3α Ser21 and GSK -3β Ser9).
PKA is a pivotal kinase in cancer, vasodilation, metabolic processes, etc. PKA signaling functions to regulate cardiac function and remodeling. It also plays a significant role in regulating metabolism, with substantial alterations in its signaling observed in obese patients.