Description
Revvity cAMP – Gs assay is a competitive immunoassay intended to measure cyclic AMP (cAMP) accumulation in cells. It enables the direct pharmacological characterization of compounds acting on Gs-coupled receptors in either adherent or suspension cells. Based on HTRF technology, the kit contains all the reagents needed to quantify cAMP, ensuring that you benefit from a complete tool for your needs.
The HTRF cAMP Gs Detection kit is best suited for measuring cAMP modulations in response to Gs-activation, leveraging an optimized combination of robustness and sensitivity.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
cAMP kits are based on a competitive format involving a specific antibody labeled with cryptate (donor) and cAMP coupled to d2 (acceptor). This enables the direct characterization of all types of compounds acting on Gs-coupled receptors in either adherent or suspension cells. Native cAMP produced by cells competes with d2-labeled cAMP for binding to monoclonal anti-cAMP Eu3+ cryptate.
Assay protocol
The protocol is simple and straightforward, with just two incubation steps: - Cell stimulation by the target compounds - cAMP detection using HTRF reagents
Validation on agonist dose response
HEK293 cells expressing the endogenous ß2 adrenergic receptor were treated with the isoproterenol agonist for 45min.
Isoproterenol was well characterized, displaying the right potency in good correlation with the literature.
Validation on antagonist dose response
HEK293 cells at 7,500 cells/well (optimal cellular concentration situated in the dynamic range of the kit) were treated with a serial concentration of isoproterenol, a beta 2 adrenergic receptor full-agonist.
The EC80-EC90 was determined to choose the optimal agonist concentration to use for the antagonist mode experiment.
Fitting the serial dilution of the antagonist allowed IC50 calculation for the compounds (potency) which was in perfect correlation with the literature.
