The second messenger cyclic GMP (cGMP) modulates cellular processes, including smooth muscle relaxation, kidney function, and inflammation, by activating effectors such as protein kinases, cGMP-dependent phosphodiesterases, and cGMP-gated ion channels.
The CatchPoint® cGMP Fluorescent Assay Kit’s high-affinity reagents are optimized for sensitivity and precision in applications where cGMP 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 cGMP Fluorescent Assay Kit
- High affinity reagents are optimized for sensitivity when cGMP 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 cGMP in samples
Figure 1: Calibration curve, 96-well assay format. Calibration curve for the CatchPoint cyclic-GMP Kit. Data was taken 60 minutes after addition of Stoplight Red substrate. The EC50 of the cGMP calibration curve was 2.2 nM (264 fmol). Minimum detectable concentration was 0.2 nM (24 fmol).
Figure 2: Atrial natriuretic peptide dose response in RFL-6 cells, 96-well assay format. A confluent monolayer of RFL-6 cells in a 12-well plate was pre-incubated for 10 minutes in Krebs Ringer bicarbonate buffer containing the non-selective phosphodiesterase inhibitor IBMX. Cells were subsequently stimulated with ANP for 15 minutes, lysed, and analyzed using the cGMP Fluorescent Assay Kit.
CatchPoint cGMP Assay Principle
The cGMP in the sample or standard competes with horseradish peroxidase (HRP)-labeled cGMP conjugate for binding sites on the anti-cGMP antibodies. In the absence of cGMP, most of the HRP-cGMP conjugate is bound to the antibody. Increasing concentrations of cGMP competitively decrease the amount of bound conjugate, thus decreasing measured HRP activity