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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. Based on our patented HTRF® technology, the cGMP kit is a highly robust assay for quantifying Guanylyl cyclase activity and phosphodiesterase monitoring.

Features

  • Homogeneous assay
  • Streamlined single-plate protocol for HTS
  • Whole cell functional assay
  • Highly sensitive assay
  • Large cGMP detection range
  • Miniaturization down to < 10 µL

Applications

  • cGMP quantification
  • Phosphodiesterase (PDE) monitoring
  • Guanylyl cyclase activity assessment
  • Receptor activation studies

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.

 

The cGMP assay requires only a single incubation following cell stimulation and can be run in under 2 hours.

 

Working range: 0.5 to 500 nM
EC50: 21 nM
Detection limit: 0.7 nM
Specificity: cGMP 100%, GMP < 0.003%,
GDP < 0.001%,
GTP < 0.003%,
AMP, ATP & cAMP < 0.001%

Biomarker cGMP standard curve

The cGMP assay is extremely sensitive and has a wide working range (0.5 - 500 nM), giving the flexibility to use a wide range of cells per well.

Figure 1 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 2 for cells stimulated with Atrial Natriuretic Peptide (ANP), a direct activator of particulate GC.

ANP dose-response curve

Figure 1: 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 2: 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.

 

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> cAMP/cGMP
PDE10 brain cAMP/cGMP
PDE11 Skeletal muscle, prostate cAMP/cGMP

 

IBMX (3-Isobutyl-1-Methylxanthine) is a non-selective inhibitor and Rolipram, 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

DescriptionCat. noProduct insertMSDS
cGMP kit - 1,000 tests62GM2PEB
cGMP kit - 20,000 tests62GM2PEC

Companion products

DescriptionCat. noProduct insertMSDS
Diluent 1 - 20 ml62DL1DDD
-
cGMP calibrator 62GMPCDA
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cGMP control 62GMPTDA
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Reconstitution buffer 2 - 13 ml62RB2RDD
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