Cell signaling via G protein-coupled receptors (GPCRs) can be assessed by monitoring the downstream effectors calcium or Cyclic AMP (cAMP). Monitoring levels of cAMP, a second messenger produced in response to activation of adenylate cyclase, is one of the most common ways to screen for agonists and antagonists of GPCRs.
The CatchPoint® cAMP Fluorescent Assay Kit’s high-affinity reagents are optimized for sensitivity and precision in applications where cAMP levels are low. A single wash step removes unbound material prior to the development step, so the assay is very resistant to interference from colored or fluorescent test compounds. The proprietary Stoplight Red substrate is used to generate a stable fluorescent readout, allowing the assay to be read in as little as 10 minutes or up to 24 hours after substrate addition.
Benefits of CatchPoint cAMP Fluorescent Assay Kit
- High affinity reagents are optimized for sensitivity when cAMP levels are low, ensuring results when other assays fail
- Robust format resists interference from colored or fluorescent compounds, improving reliability of results
- Rapid signal development (< 10 min) and no termination step give users a flexible read time that is compatible with automation and high-throughput screening
- Quantitative method allows accurate determination of cAMP in samples
Figure 1: The calibration curve for the CatchPoint® cAMP Assay Kit was generated on a SpectraMax® M5 Multi-Mode Microplate Reader. Data were taken 24 hours after addition of Stoplight Red substrate. The EC50 of the cAMP calibration curve was 5.1 nM.
Figure 2:10,000 cells were stimulated in a volume of 20 μL for 15 minutes, then lysed and analyzed using the CatchPoint® cAMP Fluorescent Assay Kit. Data were generated on a SpectraMax® M5 Multi-Mode Microplate Reader.
CatchPoint cAMP Assay Principle
The cAMP in the sample or standard competes with horseradish peroxidase (HRP)-labeled cAMP conjugate for binding sites on the anti-cAMP antibodies. In the absence of cAMP, most of the HRP-cAMP conjugate is bound to the antibody. Increasing concentrations of cAMP competitively decrease the amount of bound conjugate, thus decreasing measured HRP activity.
For more details, please refer to the CatchPoint cAMP Assay Kit product insert, which may currently be accessed through the Knowledge Base.