Cyclic GMP (cGMP) ELISA Kit

Details for Product No. ABIN2345111, Supplier: Log in to see
Antigen
Reactivity
Chemical
Alternatives
Kits with alternative reactivity to:
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Method Type
Competition ELISA
Application
ELISA
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Purpose An anti-Rabbit IgG polyclonal coating antibody is adsorbed onto a microtiter plate. Cyclic GMP present in the sample or standard competes with Peroxidase cGMP Tracer for plate binding, in the presence of Rabbit Anti-cGMP Polyclonal Antibody. Following incubation and wash steps, any Peroxidase cGMP Tracer bound to the plate is detected with addition of Chemiluminescent Reagent. The light product formed is inversely proportional to the amount of cGMP present in the sample. This reaction is then measured in a plate luminometer. A standard curve is prepared from cGMP Standard and sample concentration is then determined. 2
Sample Type Plasma, Urine
Analytical Method Quantitative
Detection Method Chemiluminescent
Sensitivity 1 pmol/mL
Characteristics cGMP ELISA Kit is a competitive enzyme immunoassay designed to measure cGMP in cell culture supernatants, plasma, serum, saliva, urine, and cell lysates. The kit selectively measures cGMP levels without any significant cross reactivities to other nucleotides or cyclic nucleotides. Samples containing low cGMP levels may be acetylated (reagents provided) for increased sensitivity. Under non-acetylated conditions, the kit has a detection range of 1 to 1000 pmol/mL cGMP, however, under acetylated conditions, the sensitivity is enhanced (approx 50X) to a detection range of 100-6250 fmol/mL.
Components
  1. Goat Anti-Rabbit Antibody Coated Plate : One strip well 96-well plate.
  2. cGMP Standard : One 200 μL vial provided at 10 mM.
  3. Rabbit Anti-cGMP Polyclonal Antibody : One 15 μL vial.
  4. Peroxidase cGMP Tracer Conjugate : One 30 μL vial.
  5. Assay Diluent : One 25 mL bottle.
  6. Lysis Buffer : One 50 mL bottle.
  7. 10X Wash Buffer : One 50 mL bottle.
  8. Triethylamine : One 2 mL amber bottle.
  9. Acetic Anhydride : One 1 mL amber bottle.
  10. Chemiluminescent Reagent A : One 6 mL amber bottle.
  11. Chemiluminescent Reagent B (Part. No. 250103): One 6 mL bottle.
Material not included
  1. Orbital plate shaker
  2. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  3. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  4. Multichannel micropipette reservoir
  5. Plate Luminometer
  6. Glass or polypropylene tubes for acetylated samples and standards
Alternative Name cGMP
Background Guanosine 3',5'-cyclic monophosphate (cGMP) is a multi-functional second messenger involved in various cellular activities in many cell and tissue types. It is converted from guanosine triphosphate (GTP) via guanylyl cyclases (GC). It has been shown that the level of cGMP is typically 10-100 fold lower than cAMP in most tissues. cGMP primarily affects cellular activities through four different pathways. These include cGMP-dependent Protein Kinases (PKG/GK), cyclic nucleotide-gated (CNG) channels, cAMP-dependent Protein Kinase (PKA), and Phosphodiesterases. PKGs (PKG I and PKG II) are serine/threonine kinases activated by cGMP. PKG I has several putative targets, many of which are involved in the regulation of smooth muscle cell (SMC) contractility. Other cGMP/PKG I substrates that affect contractility include the myosin-binding subunit (MBS) of MLC Phosphatase and the small GTPase Rho. In addition to its role in smooth muscle relaxation, cGMP/PKG I may also regulate cell survival, proliferation, axon guidance, synaptic plasticity, inflammation, and angiogenesis.
Research Area Signaling
Comment

  • Sensitivity as low as 1 pmol/mL
  • Suitable for use with cell and tissue lysates, urine, plasma, or culture medium
  • Convenient strip-well plate format

Plate Uncoated
Reagent Preparation
  • 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
  • Rabbit Anti-cGMP Polyclonal Antibody: Immediately before use dilute the Rabbit Anti-cGMP Antibody 1:500 with Assay Diluent. Do not store diluted solutions. 3
  • Peroxidase cGMP Tracer Conjugate: Immediately before use dilute the Peroxidase cGMP Tracer Conjugate 1:100 with Assay Diluent. Do not store diluted solutions.
  • Chemiluminescent Reagent: Immediately before use, mix equal volumes of Chemiluminescent Reagent A with Chemiluminescent Reagent B. Do not store diluted solutions.
  • Acetylation Reagent: Preparation of the Acetylation Reagent should be done in glass tubes and in a fume hood. The Acetylation Reagent is made by mixing Acetic Anhydride with Triethylamine at a 1:2 ratio (example: 0.5 mL Acetic Anhydride + 1 mL Triethylamine). Use the reagent within 60 minutes of preparation. Caution: The components of this reagent are known to be caustic, corrosive, flammable, and lachrymators. Use appropriate protection when handling. Preparation of cGMP Standards (Non-Acetylated Version) 1. Thaw the cGMP Standard at room temperature and mix thoroughly by pipetting (cGMP can precipitate when frozen but will redissolve when mixed well). Freshly prepare a dilution series of cGMP Standard in the concentration range of 1 mM - 1 nM by diluting the cGMP Standard in Lysis Buffer (Table 1). Lysis Buffer cGMP Standard Tubes cGMP Standard (μL) (μL) Concentration 1 40 360 1 mM 2 20 of Tube #1 180 100 μM 3 20 of Tube #2 180 10 μM 4 20 of Tube #3 180 1 μM 5 20 of Tube #4 180 100 nM 6 20 of Tube #5 180 10 nM 7 20 of Tube #6 180 1 nM 8 0 180 0 Table 1.
Assay Procedure
  1. Prepare and mix all reagents thoroughly before use.
  2. Each cGMP sample, cGMP Standard, and blank should be assayed in duplicate. Note: cGMP samples must be compared with corresponding standards (i.e. acetylated samples compared with acetylated standards, non-acetylated samples with non-acetylated standards).
  3. Add 50 μL of cGMP sample or standard (acetylated or non-acetylated) to the Goat Anti-Rabbit Antibody Coated Plate.
  4. Add 25 μL of diluted Peroxidase cGMP Tracer Conjugate (see Preparation of Reagents Section) to each tested well.
  5. Add 50 μL of diluted Rabbit Anti-cGMP Polyclonal Antibody (see Preparation of Reagents Section) to each tested well.
  6. Cover with a Plate Cover and incubate at room temperature for 30 minutes with shaking.
  7. Remove Plate Cover and empty wells. Wash microwell strips 5 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
  8. Add 100 μL of Chemiluminescent Reagent (see Preparation of Reagents Section) to each well, including the blank wells. Incubate at room temperature for 5 minutes on an orbital shaker.
  9. Read the luminescence of each microwell on a plate luminometer. 6
Restrictions For Research Use only
Storage 4 °C/-20 °C
Storage Comment Store kit components at 4°C. For longer term use, store the Rabbit Anti-cGMP Polyclonal Antibody at -20°C.
Product cited in: Engels, Elting, Braun, Bendix, Herz, Felderhoff-Müser, Dzietko: "Sildenafil Enhances Quantity of Immature Neurons and Promotes Functional Recovery in the Developing Ischemic Mouse Brain." in: Developmental neuroscience, 2017 (PubMed).

Yuan, Peng, Khan, Nanduri, Singh, Vasavda, Semenza, Kumar, Snyder, Prabhakar: "H2S production by reactive oxygen species in the carotid body triggers hypertension in a rodent model of sleep apnea." in: Science signaling, Vol. 9, Issue 441, pp. ra80, 2016 (PubMed).

Yuan, Vasavda, Peng, Makarenko, Raghuraman, Nanduri, Gadalla, Semenza, Kumar, Snyder, Prabhakar: "Protein kinase G-regulated production of H2S governs oxygen sensing." in: Science signaling, Vol. 8, Issue 373, pp. ra37, 2015 (PubMed).