Protein Kinase A (PKA) Colorimetric Activity Kit

Research Areas

Documentation

Specifications

• 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
• Description

  Assay Principle: 

  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.

  Background:

  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.