Membrane-Based Assay with cAMP
HiRange kit
Cisbio Bioassays's cAMP HiRange kit is designed to address either the use of cell-based and cellular membranes-assays in GPCR activation and Adenylate Cyclase activity screening. The cAMP HiRange assay is highly robust and produces quality results while eliminating the need for a continuous supply of living cells.
- Simple and rapid assay protocol
- Small quantity of membrane required per assay
- Signal to background ratios on the calibration curve >34
- Amenable to automation
- Gs or Gi coupled receptor activation
- Agonist / antagonist screening
- Phosphodiesterase (PDE) monitoring
- Adenylate cyclase activity assessment
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 followed by the addition of HTRF detection reagents. Signals at 620 and 665nm are detected after a 1 hour incubation.
Product Specifications:
| S/B | 34 |
| EC50 | 22.8 nM |
| Assay range | 0.17-2,2800 nM cAMP |
Figure 1: The calibration curve was obtained by pre-mixing the assay calibrators with cAMP-d2 and then various concentrations of cAMP added (0.17nM to 2,800nM). The assay was run using the recommended kit protocol.
Assay Performance:
The cAMP HiRange kit is useful for monitoring Gi/s receptor activation as well as adenylate cyclase screening. This assay has specifically been optimized for use with cellular and membranes preparation. For membrane-based assay 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.
Figure 2: 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.
Ordering Info:
cAMP Cell-based Assay Reagents:
| Description | Quantity* | Cat no. | Product Insert |
| cAMP HiRange kit | 1,000 tests | 62AM6PEB | |
| cAMP HiRange kit | 20,000 tests | 62AM6PEC | |
| cAMP HiRange kit | 100,000 tests | 62AM6PEJ | |
| * based on 20 µl assay volume | |||
Companion Products:
| Description | Cat no. |
| cAMP HiRange calibrator | 62AM6CDA |
| cAMP HiRange control | 62AM6TDA |
| Diluent for standard curve prep. | 62DL1DDD |
For more information or To Place an Order:
U.S. and Canada
1-888-963-4567
Europe and other countries
+33(0)466 796 705
Japan
+81-3-5510-2932
