( e basis of molecular size. The tracer-a...)cAMP DataFile: Homogenous Cyclic AMP detection using High Efficiency Fluorescence Polarization

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.

Kit Contents
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.

Assay Conditions
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.

Applications
Measurement of adenylyl cyclase activity¹
Monitoring phosphodiesterase activity²

Assay Performance
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.

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