cGMP Assay
Cyclic GMP (Guanosine 3',5'-cyclic monophosphate) is a major second messenger that mediates cell activities by activating protein effectors such as protein kinase G (PKG), cGMP-regulated phosphodiesterases (PDEs), and cGMP-gated channels. Cellular cGMP is synthesized by soluble Guanylyl Cyclases and particulate Guanylyl cyclase, triggered by nitric oxide and atrial natriuretic peptide (ANP) binding respectively. Key processes such as smooth muscle relaxation, kidney function and inflammatory responses are known to be regulated through cGMP signaling. Cisbio Bioassays developed a highly robust HTRF-based assay for the quantification of cGMP to address the needs of discovery researchers investigating cGMP.
- Streamlined single-plate protocol for HTS
- Whole cell functional assay
- Highly sensitive assay
- Large cGMP detection range
- Miniaturization down to < 10 µL
- Proven cGMP assay specificity
- Developed with the new HTRF acceptor d2
- cGMP quantification
- Phosphodiesterase (PDE) monitoring
- Guanylyl cyclase activity assessment
- Receptor activation studies
Assay Principle:
The cGMP assay is a competitive immunoassay that uses Eu3+ cryptate-labeled anti-cGMP and d2-labeled cGMP. The assay is compatible with cell supernatants, whole cells or frozen cells. The basic protocol has two phases, cell stimulation and cGMP detection using the HTRF labeled reagents. The assay requires only a single incubation following cell stimulation and can be run in under 2 hours.
Product Specifications:
| Detection Limit: | 0.5 nM cGMP |
| Working Range: | 0.5 to 500 nM |
| SB: | >15 |
| Assay Specificity: | |
| cGMP | 100% |
| GMP | < 0.003% |
| GDP | < 0.001% |
| GTP | < 0.003% |
| AMP, ATP & cAMP | < 0.001% |
Figure 1: The cGMP assay requires only a single incubation following cell stimulation and can be run in under 2 hours.
Assay Performance:
The cGMP assay is extremely sensitive and has a large working range (0.5 - 500 nM) allowing the flexibility to use a wide range of cells per well. Figure 2 shows the results of assays performed using 300 and 20,000 cells per well and stimulated with S-nitroso-N-acetylpenicil amine (SNAP), a direct activator of soluble Guanylate Cyclase (GC) enzyme. Statistically significant quantities of cGMP were detected from as few as 300 cells. A typical dose response curve is shown in Figure 3 for cells stimulated with Atrial Natriuretic Peptide (ANP), a direct activator of particulate GC.
Figure 2: 300 to 20,000 RFL-6 rat lung fibroblasts were plated per well in a low volume 384-well microplate. Cells were incubated either in the presence or absence of SNAP and cGMP quantified using the HTRF cGMP kit following the standard assay protocol.
Figure 3: 10,000 RFL-6 rat lung fibroblasts were plated per well in a low volume 384-well microplate and stimulated with increasing concentrations of ANP. cGMP levels were calculated using the HTRF cGMP kit following the standard assay protocol.
Phosphodiesterase assays using HTRF technology :
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of cellular cGMP and cAMP upon activation. More than 30 PDEs have been identified and can be classified into at least 11 families of isoenzymes, PDEs 1-11 (see table).
The discovery of sildenafil (Viagra®, PDE5 inhibitor), and of several PDE4 inhibitors as anti-inflammatory drugs for treating asthma contributed to the growing interest in PDEs as drug targets. A study presented at SBS 2004 described the development of a rapid and robust PDE assay using HTRF cAMP and cGMP kits. Figure 4 summarizes a study conducted using the HTRF cGMP assay and known inhibitors of PDE4 such as IBMX, Rolipram, and Zaprinast.
| Gene family | Major tissue expression | Substrate |
| PDE2 | Adrenal cortex, brain, heart | cAMP/cGMP |
| PDE3 | Heart, adipose tissue, pancreas, platelets | cAMP/cGMP |
| PDE4 | Many tissues | cAMP |
| PDE5 | Lung, platelets, smooth muscle, corpus cavernosum | cGMP |
| PDE6 | Rod and cone photoreceptor outer segments | cGMP |
| PDE7 | Skeletal muscle, T-cells, B-cells | cAMP |
| PDE8 | Testis, liver, thyroid | cGMP |
| PDE9 | Kidney/td> | cGMP |
| PDE10 | brain | cAMP/cGMP |
| PDE11 | Skeletal muscle, prostate | cAMP/cGMP |
Figure 4: IBMX (3-Isobutyl-1-Methylxanthine) is a non-selective inhibitor and Rolipram, is a selective inhibitor of PDE4. The PDE4 concentration is 5 ng/ml and cAMP substrate is used at a final concentration of 44 nM
Ordering Info:
cGMP Assay Reagents:
| Description | Quantity* | Cat no. | Product Insert | MSDS |
| cGMP kit | 1,000 tests | 62GM2PEB | ||
| cGMP bulk kit | 20,000 tests | 62GM2PEC | ||
| *based on 20 µl assay volume | ||||
Companion Products:
| Description | Cat no. |
| cGMP calibrator | 62GMPCDA |
| cGMP control | 62GMPTDA |
For more information or To Place an Order:
Europe and other countries
+33(0)466 796 705
