Membrane-based assays with cAMP HiRange kit

Assay Principle

The cAMP HiRange assay is a competitive immunoassay which uses Eu3+ cryptate-labeled MAb anti-cAMP and d2-labeled cAMP. Simply dispense membrane preparations or whole cells and sample compounds. Incubate for 30 minutes, then add the HTRF® detection reagents. Signals at 620 and 665nm are detected after a 1 hour incubation.

Product Specifications

The calibration curve was obtained by pre-mixing the assay calibrators with cAMP-d2, and then various concentrations of cAMP were added (0.17nM to 2,800nM). The assay was run using the recommended kit protocol.

Assay Performances

The cAMP HiRange kit is useful for monitoring Gi/s receptor activation, as well as for adenylate cyclase screening. This assay has been specifically optimized for use with cellular and membrane preparations. For membrane-based assays, the stimulation buffer contains ATP and GTP*. The robust nature of HTRF® technology allows high quality results despite the complex nature of membrane preparations. Assays can easily be run in a 384-well microplate for high throughput screening (Figure 2). Stimulation buffer for membrane-based assay: HEPES pH 7.4 33mM, MgCl2 2mM, tween 0.1%, ATP 500µM, GTP 2µM and IBMX (Phosphodiesterase inhibitor) 500µM.

Membranes were prepared from CHO-K1 cell lines stably expressing Histamine H2 (Gs-coupled) GPCR. 2 to 8 µg H2 membranes were dispensed per well in 384-well low volume plates. H2 membranes were stimulated with increasing concentrations of Amthamine (agonist). cAMP membrane assays were performed using the standard kit protocol in a 20 µl final assay volume.

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