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HTRF cGMP Detection Kit, 500 Assay Points

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

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Part Number: 62GM2PEG
Unit Size: 500 Assay Points
Part Number: 62GM2PEH
Unit Size: 10,000 Assay Points
  • 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.

  • Assay Points
    500
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Shipping Conditions
    Shipped Ambient
    Therapeutic Area
    Cardiovascular
    Unit Size
    500 Assay Points
  • 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.

    biomarkers-how-it-works-assay-principle-cgmp
    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.

    biomarkers-how-it-works-assay-protocol-cgmp

     

  • 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%
    specifications-cgmp

     

  • 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.

    assay-validation-cgmp

    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.

    assay-validation-cgmp
    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.

    assay-validation-cgmp
    assay-validation-cgmp
    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

    assay-validation-cgmp

     

  • 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.

    biomarkers-pathway-cgmp-kit