Description
Key facts
Detection method:Colorimetric,
Sample types:Purified protein, Suspension cells, Tissue Extracts, Adherent cells,
Assay type:Enzyme activity,
Assay time:4h 30m,
Assay Platform:Microplate reader
Product details:
PKA Kinase Activity Kit (ab139435) is a non-radioactive assay providing a safe, rapid and reliable method for the screening of inhibitors or activators of PKA and for quantitating the activity of PKA in purified or partially purified enzyme preparations. This kit is based on a solid phase enzyme-linked immuno-absorbent assay (ELISA) that uses a specific synthetic peptide as a substrate for PKA and a polyclonal antibody that recognizes the phosphorylated form of the substrate.
PKA activity assay principle
In the assay, a synthetic peptide is used as PKA specific substrate. The substrate, which is pre-coated on the microplate provided, is phosphorylated by the PKA present in the sample after addition of ATP. The phosphorylated substrate is then recognized by a polyclonal antibody that recognizes only the phospho-substrate.
The phospho-specific antibody is subsequently recognized by an HRP conjugated secondary antibody. The assay is finally developed with TMB substrate and color develops in proportion to the PKA activity existing in the sample.
The color development is stopped with acid stop solution and the intensity of the color is measured in a microplate reader at OD 450 nm.
The assay is designed for the analysis of PKA activity in the solution phase. This kit gives you the ability to an end-point or kinetic assay read-out in a convenient 96-well plate based assay and offers easy sample handling protocols.
The kit offers the following advantages:
1. Safe - non-radioactive measurement of kinase activity
2. Flexible - kinetic and end-point options available
3. Fast - results in < 4.5 hours
4. Efficient - only 30 μl diluted sample needed per well
Properties and Storage Information:
Shipped at conditions-MULTI, Appropriate short-term storage conditions-Multi, Appropriate long-term storage conditions-Multi, Storage information-Please refer to protocols
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Protein kinase A (PKA) also known as cAMP-dependent protein kinase plays an important role in cellular signaling. It is composed of a regulatory and a catalytic subunit having a molecular mass around 150 kDa. PKA is expressed widely in tissues such as the heart brain and skeletal muscle. The activation of PKA occurs when cyclic adenosine monophosphate (cAMP) binds to its regulatory subunit leading to the release of the catalytic subunit which then phosphorylates various target proteins.
Biological function summary
PKA acts as a significant regulator of gene expression and cellular metabolism. It functions within the PKA signaling complex which includes A-kinase anchoring proteins (AKAPs) that localize PKA to specific areas in the cell aiding in precise function. PKA modulates processes such as lipolysis glycogen metabolism and transcription. Through these activities it contributes to cellular responses to hormonal changes by phosphorylating key substrates.
Pathways
PKA activation integrates into the cAMP signaling pathway an essential regulatory pathway influencing a broad range of physiological responses. This pathway connects PKA with the downstream effectors such as CREB and other transcription factors. Moreover PKA is involved in the MAPK pathway where it interacts with proteins like MEK and ERK orchestrating responses to growth factors. These pathways demonstrate PKA's role in integrating extracellular signals into cellular actions.
Dysregulation of PKA activity associates with conditions like Cushing's syndrome and cardiac hypertrophy. In Cushing's syndrome mutations affecting the regulatory subunit of PKA can lead to unregulated hormone activity. Cardiac hypertrophy involves altered PKA pathway signaling affecting heart muscle growth and function. PKA interacts with proteins such as phospholamban in cardiac disorders impacting calcium handling and heart contractility.
