cAMP Gs dynamic kit
Optimal performance when applied to Gs activation or inhibition
cGMP kit HTRF®
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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No-wash
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All inclusive kit
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High sensitivity
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.
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.
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% |
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.
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.
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.
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
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.
Product Insert cGMP Kit / 62GM2PEG-62GM2PEH
62GM2PEG-62GM2PEH - Product Insert
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
Cardiovascular Research With HTRF Assays
Learn how peer researchers tackle cardiovascular research with HTRF assays - Application Notes
Plate Reader Requirement
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