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
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.
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 e basis of molecular size. The tracer-antibody complex rotates more slowly than free tracer so that the fluorescence appears polarized. Displacement of the tracer by the analyte from the antibody results in a decrease in polarization. Free tracer is small, spins rapidly relative to the fluorescence lifetime and thus emits light that is detected as having low polarization. HEFP does not require physical separation steps or wash steps.
The reagents have been optimized to provide a sensitive, robust and rapid homogeneous assay. Individual assays of cell lysates or of purified adenylyl cyclase will require optimization and validation based upon the requirements of the individual test systems.
The kit contains anti-cAMP antibody (100X), tracer (fluorescein-labeled cAMP, 100X), calibrator cAMP (500 M) and HEFP Assay Buffer. When used as suggested, each kit provides sufficient reagents for 800 40-L assays.
Samples or calibrators containing cAMP are mixed with fluorescein-labeled tracer and anti-cAMP antibody in microplate wells and analyzed after 2 hours.
The kit is designed for standard 384-well microplates with a 40 L sample volume. It can be scaled down to an 8 L sample volume, with the same performance, using HE microplates in any instrument in the Criterion family.
Measurement of adenylyl cyclase activity1
Monitoring phosphodiesterase activity2
Calibration curve: A calibration curve was performed in 384-well microplates with a final volume of 40 L. Background signals (buffer only or antibody only) were appropriately subtracted and net polarization calculated. The minimum detectable concentration was 0.1 pmol.
Measuring adenylyl cyclase activity: Cultured T-47D cells were treated for 15 min with either buffer (control), forskolin, isoproternol, or isoproterenol and propranolol. Formation of cAMP was determined with the cAMP Assay Kit. The assay was performed in 384-well microplates in a final volume of 40 L. Each treatment group was performed in triplicate.
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1 Chedeville, A. et al. FEBS Letters 319:171176 1993
2 Chan, S.C. and Hanifan, J.M. S. J. Clin. Med. 121:4451 1993