The cGMP kit is designed for the accurate quantitative measurement of Cyclic GMP, a major second messenger that mediates various cell activities.
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  • All inclusive kit All inclusive kit
  • High sensitivity High sensitivity
The cGMP kit is designed for the accurate quantitative measurement of Cyclic GMP, a major second messenger that mediates various cell activities.
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Overview

The cGMP assay kit accurately measures Cyclic GMP produced by cells. The assay can be used to screen molecules that modulate the Phosphodiesterase (PDE) activity. cGMP is a major second messenger that mediates cell activities by activating various protein effectors. Cellular cGMP is synthesized by soluble guanylyl cyclases and particulate guanylyl cyclase, triggered by nitric oxide and atrial natriuretic peptide (ANP) binding respectively. The cGMP kit offers superior benefits over traditional ELISA readouts with the ease-of-use and high sensitivity of homogeneous TR-FRET assays.

Benefits

  • PHOSPHODIESTERASE (PDE) MONITORING
  • GC ACTIVITY ASSESSMENT
  • RECEPTOR ACTIVATION STUDIES

Assay principle

The kit is based on a competitive format involving a specific antibody labelled with Cryptate (donor) and cGMP coupled to d2 (acceptor). cGMP produced by cells competes with d2-labelled cGMP for binding to monoclonal anti-cGMP Eu Cryptate.
cGMP assay principle

Assay protocol

The cGMP assay features a streamlined protocol with only two incubation steps:

- Cell stimulation by target compounds

- cGMP detection using HTRF reagents.

This protocol requires only a single, one-hour incubation period following cell stimulation.

cGMP assay protocol

Specifications

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%
cGMP assay standard curve

Cell dependent cGMP production & ANP titration

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 amines (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.
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.
cGMP production on fresh and frozen cells
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.
ANP dose response curve

Phosphodiesterase

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

Phosphodiesterase inhibitor testing

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.
PDE inhibitor dose response
PDE inhibitor dose response

Dose-response analysis of IW-1973 over cGMP and pVASP Ser 2 39

Increases in IW-1973 increases both cGMP production and VASP phosphorylation. IW-1973 is a sGC stimulator. cGMP production and VASP phosphorylation were measured using the HTRF assays.


Adapted from Tobin J.V, et al (2018). Pharmacological characterization of IW-1973, a novel soluble guanylate cyclase stimulator with extensive tissue distribution, anti-hypertensive, anti- inflammatory,and anti-fibrotic effects in preclinical models of disease. J Pharmacol Exp Ther 365(3):664-675

Dose-response analysis of IW-1973 over cGMP and pVASP Ser 2 39

Nitric Oxide signaling pathway

Nitric Oxide is a key effector protein that - among other roles - is involved in cardiovascular hemostasis and vascular relaxation. In the event of a vascular stress, NO is synthesized at its location. It binds to sGC on the environing tissues, triggering the downstream decrease of Ca2+ and increase of cGMP, which in turn alters the activity of a collection of protein kinase G, cyclic nucleotide-gated ion channels and phosphodiesterases, leading to the vasodilatation of nearby vessels, blood flow increase and lowered tension.

Nitric Oxide simplified signaling pathway
Product Insert cGMP Kit / 62GM2PEG-62GM2PEH

62GM2PEG-62GM2PEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Safety Data Sheet cGMP Kit / 62GM2PEG

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