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| Product Name | Nitric Oxide Detection Assay Kit (Griess Reagent Method) |
| Catalog No. | CMTR-HMM-0064 |
| Description | A colorimetric assay for the quantitative detection of nitrite (NO2-), the stable and non-volatile oxidation product of nitric oxide, using the diazotization-based Griess reaction. Nitrite reacts with sulfanilamide to form a diazonium salt that couples with N-(1-naphthyl)ethylenediamine to produce a pink azo dye with absorbance at 540 nm. |
| Intended Use | Measurement of nitric oxide production in cell culture supernatants, tissue homogenates, and biological fluids as an indicator of nitric oxide synthase activity in inflammation, vascular biology, neurobiology, and immunology research. |
| Principle / Technology | Under acidic conditions, nitrite reacts with sulfanilamide to generate a diazonium intermediate; this diazonium ion then couples to N-(1-naphthyl)ethylenediamine dihydrochloride to produce a stable pink azo chromophore (Griess reaction product) with maximal absorbance at 540 nm; each mole of nitrite generates one mole of azo dye; nitrate (NO3-) in the sample does not react directly but can be converted to nitrite using nitrate reductase for total NO measurement. |
| Detection Method | Colorimetric absorbance at 540 nm (range 520–550 nm) using a microplate spectrophotometer or standard spectrophotometer |
| Sample Type | Cell culture supernatant (from macrophages, endothelial cells, neurons stimulated with LPS, IFN-γ, or other NOS inducers), serum and plasma, urine, cerebrospinal fluid, tissue homogenate supernatant |
| Performance Range / Specifications | Nitrite standard curve: 0.5–100 μM (linear 1–100 μM); incubation time: 10–15 minutes at room temperature; azo dye stable for approximately 30 minutes; absorbance range 0.05–1.5 OD at 540 nm |
| Sensitivity / LOD | Detection limit: approximately 0.5 μM nitrite in aqueous solution with 100 μL sample volume in 96-well microplate format |
| Specificity | Griess reaction is highly specific for nitrite (NO2-); nitrate (NO3-), nitric oxide (NO), and peroxynitrite (ONOO-) do not produce color directly; sample components such as phenol red (culture media), serum proteins, and common buffer components do not interfere at standard Griess reaction volumes; azide and reducing agents may cause interference |
| Reaction Conditions / Protocol | Deproteinize samples if needed (serum, plasma) using zinc sulfate and sodium hydroxide precipitation; add 50 μL sample or nitrite standard to microplate wells; add 50 μL Griess Reagent A (1% sulfanilamide in 5% phosphoric acid); incubate 5–10 minutes at room temperature protected from light; add 50 μL Griess Reagent B (0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in water); incubate 5–10 minutes until pink color develops; measure absorbance at 540 nm; for total NO (nitrite + nitrate), pre-treat samples with nitrate reductase and NADPH cofactor for 30 minutes at 37°C before Griess reaction |
| Components / Formulation | Griess Reagent A (sulfanilamide, 1% w/v in 5% phosphoric acid), Griess Reagent B (N-(1-naphthyl)ethylenediamine dihydrochloride, 0.1% w/v), Nitrite Standard (sodium nitrite, 100 μM or 1 mM ready-to-use), Nitrate Reductase enzyme and NADPH cofactor for total NO measurement (optional component), Assay Buffer |
| Storage Conditions | Griess Reagents A and B: 2–8°C protected from light, stable for 12 months; Nitrite Standard: 2–8°C for 6 months or -20°C for 12 months; Nitrate Reductase (if included): -20°C |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 500 assays (96-well format), 1,000 assays (96-well format) |
| Product Form | Liquid ready-to-use reagents with optional lyophilized nitrate reductase component |
| Quality Control | Each lot standardized against 0–100 μM sodium nitrite reference solution; linear calibration curve (R2 ≥0.999); Griess reagent blank absorbance <0.03 at 540 nm; nitrate-to-nitrite conversion efficiency >95% for total NO measurement component |
| Key Features | Simple and rapid protocol without incubation at elevated temperature; highly cited methodology compatible with published data comparison; adaptable to both nitrite-only and total NO/nitrate measurement; stable color development suitable for batch processing; very low reagent cost per test |
For research use only, not for clinical use.
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