Nitric Oxide Assay Kit

Details for Product No. ABIN1000244
Antigen
Application
Biochemical Assay (BCA)
Options
Sample Type Cell Lysate, Food, Plasma, Serum, Tissue Lysate, Urine
Specificity 0.6 μM
Characteristics Sensitive and accurate. Detection range 0.6 - 200 µM in 96-well plate.
Rapid and reliable. Using an optimized VCl3 reagent, the time required for reduction of NO3 - toNO2 - is 10 min at 60°C.
Simple and high-throughput. The procedure involves mixing sample with three reagents, incubation for 10 min at 60°C and reading the optical density. Can be readily automated to measure thousands of samples per day.
Components Reagent A: 12 mL. Reagent B: 500 µL. Reagent C: 12 mL. NaOH: 1 mL. ZnSO4: 1 mL. Standard: 1 mL.
Material not included Pipetting devices, eppendorf tubes, eppendorf centrifuge, clear, flat bottomed 96 well plates or cuvettes, plate reader or spectrophotometer and heat block or hot water bath (optional).
Target Name (Antigen)
Alternative Name Nitric Oxide
Background Quantitative determination of nitric oxide by colorimetric (540nm) method.
Procedure: 40 min.

Nitric oxide (NO) is a reactive radical that plays an important role in many key physiological functions. NO, an oxidation product of arginine by nitric oxide synthase, is involved in host defense and development, activation of regulatory proteins and direct covalent interaction with functional biomolecules. Simple, direct and automation-ready procedures for measuring NO are becoming popular in Research and Drug Discovery. Since NO is oxidized to nitrite and nitrate, it is common practice to quantitate total NO2- /NO3- as a measure for NO level. This Nitric Oxide Assay Kit is designed to accurately measure NO production following reduction of nitrate to nitrite using improved Griess method. The procedure is simple and the time required for sample pretreatment and assay is reduced to as short as 30 min.
Application Notes Direct Assays: NO in plasma, serum, urine, tissue/cells and foods.
Drug Discovery/Pharmacology: effects of drugs on NO metabolism.
Comment

Antioxidants and nucleophiles (e.g. beta-mercaptoethanol, glutathione, dithiothreitol and cysteine) may interfere with this assay. Avoid using these compounds during sample preparation.

Protocol Sample treatment: tissue or cell samples are homogenized in 1 x PBS (pH 7.4). Centrifuge at 10,000g or higher at 4°C. Use supernatant for NO assay. Samples that need deproteination include serum, plasma, whole blood, cell culture media containing FBS, tissue or cell lysates. Urine and saliva do not need deproteination. Deproteination. Mix 150 µL sample with 8 µL ZnSO4 in1.5-mL tubes. Vortex and then add 8 µL NaOH, votex again and centrifuge 10 min at 14,000 rpm. Transfer 100 µL of the clear supernatant to a clean tube. Note: If samples need to be deproteinated, 150 µL of each standard should be prepared and also treated with ZnSO4 and NaOH to eliminate the need for a dilution factor.

Procedure using 96-well plate:
1. Standards. Prepare 500 µL 100 µM Premix by mixing 50 µL 1.0 mM Standard and 450 µL distilled water.
2. Reaction. Add 100 µL of each sample to separate, labeled eppendorf tubes. (We recommend that samples be measured in at least duplicate). Immediately prior to starting the reaction, prepare enough Working Reagent (WR) for all samples and standards by mixing per reaction tube: 100 µL Reagent A, 4 µL Reagent B and 100 µL Reagent C. Add 200 µL of the WR to each sample and standard tube and incubate for 10 min at 60°C. (Alternatively, the reaction can be run at 37°C for 60 min or RT for 150 min.)
3. Measurement. Briefly centrifuge the reaction tubes to pellet any condensation and transfer 250 µL of each reaction to separate wells in a 96 well plate. Read OD at 500-570nm (peak 540 nm).

Procedure using Cuvette: Prepare standards and samples as described for the 96-well procedure except quadruple (4x) the volumes. After the reaction, transfer 1 mL to a cuvette. Measure OD540nm in the cuvette.
Calculation of Results

Subtract blank OD (Std 4) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting.
Conversions: 1 mg/dL NO equals 333 µM, 0.001% or 10 ppm.

Restrictions For Research Use only
Storage 4 °C
Supplier Images
Biochemical Assay (BCA) image for Nitric Oxide Assay Kit (ABIN1000244) Nitric Oxide Assay Kit
Product cited in: Zeng, Peng, Monie, Yang, Pang, Hung, Wu: "Control of cervicovaginal HPV-16 E7-expressing tumors by the combination of therapeutic HPV vaccination and vascular disrupting agents." in: Human gene therapy, Vol. 22, Issue 7, pp. 809-19, 2011 (PubMed).

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Alonso, Boittin, Bény, Haefliger: "Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta." in: American journal of physiology. Heart and circulatory physiology, Vol. 299, Issue 5, pp. H1365-73, 2010 (PubMed).

Lipari, Garcia, Zhao, Thrift, Vaidya, Rodriguez: "Nitric oxide metabolite production in the human preimplantation embryo and successful blastocyst formation." in: Fertility and sterility, Vol. 91, Issue 4 Suppl, pp. 1316-8, 2009 (PubMed).

Neuschmelting, Marbacher, Fathi, Jakob, Fandino: "Elevated level of endothelin-1 in cerebrospinal fluid and lack of nitric oxide in basilar arterial plasma associated with cerebral vasospasm after subarachnoid haemorrhage in rabbits." in: Acta neurochirurgica, Vol. 151, Issue 7, pp. 795-801; discussion 801-2, 2009 (PubMed).

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Levy, Valero, Espina, Añez, Arias, Mosquera: "Increment of interleukin 6, tumour necrosis factor alpha, nitric oxide, C-reactive protein and apoptosis in dengue." in: Transactions of the Royal Society of Tropical Medicine and Hygiene, Vol. 104, Issue 1, pp. 16-23, 2009 (PubMed).

Wang, Zhang, Lasbury, Liao, Durant, Tschang, Lee: "Decreased inflammatory response in Toll-like receptor 2 knockout mice is associated with exacerbated Pneumocystis pneumonia." in: Microbes and infection / Institut Pasteur, Vol. 10, Issue 4, pp. 334-41, 2008 (PubMed).

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Smith, White, Smith, Bowlin: "In vitro evaluations of innate and acquired immune responses to electrospun polydioxanone-elastin blends." in: Biomaterials, Vol. 30, Issue 2, pp. 149-59, 2008 (PubMed).

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Hasegawa, Wakino, Tatematsu, Yoshioka, Homma, Sugano, Kimoto, Hayashi, Itoh: "Role of asymmetric dimethylarginine in vascular injury in transgenic mice overexpressing dimethylarginie dimethylaminohydrolase 2." in: Circulation research, Vol. 101, Issue 2, pp. e2-10, 2007 (PubMed).

Background publications Bulau, Zakrzewicz, Kitowska, Leiper, Gunther, Grimminger, Eickelberg: "Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA." in: American journal of physiology. Lung cellular and molecular physiology, Vol. 292, Issue 1, pp. L18-24, 2007 (PubMed).

Bolander: "The compartmentalization of prolactin signaling in the mouse mammary gland." in: Molecular and cellular endocrinology, Vol. 245, Issue 1-2, pp. 105-10, 2006 (PubMed).

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