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cAMP DataFile: Homogenous Cyclic AMP detection using High Efficiency Fluorescence Polarization

A Sensitive, Robust, Homogenous, and Rapid Assay
Uses High Efficiency Fluorescence Polarization (HEFP) detection method
Detects pmol amounts of cAMP
Optimized for cell extract and enzyme preparations
Requires no separation or wash steps
Available in a kit and in bulk packaging


Introduction
Cyclic adenosine monophosphate (cAMP) is a second messenger involved in the transduction of extracellular signals. The signal is initiated by the binding of a ligand to a G-protein coupled receptor followed by a series of enzyme mediated steps leading to the activation of the enzyme adenylyl cyclase. Adenylyl cyclase converts ATP into cyclic AMP that in turn activates protein kinases. Protein kinase activity is correlated to the intracellular concentration of cyclic AMP. The level of cyclic AMP is reduced by phosphodiesterase. Assays that determine the activities of these cellular regulators are important tools for high-throughput screening laboratories.


Intended Use
The cAMP Assay Kit is designed to determine concentrations of cyclic AMP in solution. The sources of cyclic AMP analyte may either be in vitro enzyme preparations or cell extracts.


Principle of the Assay
The assay is an antibody-mediated competitive binding reaction with a High Efficiency Fluorescence Polarization (HEFP) read out. Cyclic AMP (analyte) from sample, calibrators, or controls competes with fluorescein-labeled cAMP (tracer) for a limited number of anti-cAMP antibodies. In the absence of analyte, a maximal amount of tracer will be bound to the antibody.

Addition of analyte competes with the tracer for the limited binding sites, so that the extent of polarization is inversely proportional to the concentration of analyte.

HEFP is a detection method that discriminates on th
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