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Stock availability
In Stock
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Kit s description
Colorimetric detection of nitrate and nitrite
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Protein target
Nitric Oxide
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Scientific context
Nitric oxide (NO) is a diffusible, transient, reactive molecule that has physiological effects in the picomolar-to-micromolar range. Acting through soluble guanylate cyclase activation, NO is an important physiological regulator of the cardiovascular, nervous, and immunological systems (1). NO is bio-available by two routes. It can be endogenously generated by constitutive or induced enzymes like Nitric Oxide Synthase or it can be orally ingested as nitrates / nitrites for rapid uptake into circulation and subsequent conversion (2). The reactive nature of nitric oxide allows it to act as a cytotoxic factor when released during an immune response by cells such as macrophages. The reactivity also allows NO to be easily converted to a toxic radical that can produce nitrosative damage to cells, organelles and molecules such as DNA. Nitrosaylation however can be a regulated post-translational modification in cell signaling (3). The balance and dynamics of the regulatory/damage facets of NO are major forces in mitochondrial signaling and dysfunction (4). NO is linked not only to coronary heart disease, endothelial dysfunctions, erectile dysfunction, and neurological disorders, but also diabetes, chronic periodontitis, autism, cancer, and assorted age-related diseases (5-9). The physical properties of Nitric Oxide make it challenging for direct detection methods. However, colorimetric methods can be applied to measure its stable break-down products nitrate (-NO3) and nitrite (-NO2) (10).
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Primary research area
Cell Signaling, Post-translational Modifications, Neuroscience
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Category
Assay Detection Kits
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Other name
Nitrogen monoxide; Nitrogen(II) oxide Detection Kit
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Brand name
StressXpress®
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Detection system
Colorimetric Assay
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Assay format
Indirect Quantitative Assay
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Main intended usage
Colorimetric assay used to quantitatively measure Nitrate and Nitrite present in a variety of samples.
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Brief protocol
The Nitric Oxide Detection Kit is designed to quantitatively measure Nitrate and Nitrite present in a variety of samples. Nitric Oxide content is derived from the sum of Nitrate (-NO3) and Nitrite (-NO2). Both Nitrate and Nitrite standards are provided to generate standard curves for the assay and all samples should be read off the appropriate standard curve. For Nitrite detection, samples are mixed with the Color Reagents A and B and incubated at room temperature for 5 minutes. The colored product is read at 550 – 570 nm. The concentration of Nitrite in the sample is calculated, after making a suitable correction for any dilution of the sample, using software available with most plate readers. Total Nitric Oxide content is measured after the sample is incubated with Nitrate Reductase and NADH. The reductase in combination with NADH reduces Nitrate to Nitrite. After a 20 minute incubation at room temperature, Color Reagents A and B are added and incubated at room temperature for 5 minutes. The colored product is read and calculated as with the Nitrite determination above. The concentration of Nitrate in the sample is calculated by subtracting the measured Nitrite concentration from the Total Nitric Oxide concentration for the sample.
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Kit contents
Clear 96 Well Plates, Nitrate Standard, Nitrite Standard, Assay Buffer, NADH Concentrate, Nitrate Reductase, Enzyme Stabilization Buffer, Color Reagent A, Color Reagent B
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Assay precision
Intra Assay Precision: Three samples were further diluted in Assay Buffer and run in replicates of 20 in an assay. The mean and precision of the calculated Nitrite or Total NO concentrations were: Sample 1 (Nitrite Concentration)- 45.1 µM, 4.4% CV, Sample 1 (Total NO Concentration)- 70.5 µM, 6.8% CV Sample 2 (Nitrite Concentration)- 73.3 µM, 9.1% CV, Sample 1 (Total NO Concentration)- 107.4 µM, 4.4% CV Sample 3 (Nitrite Concentration)- 132.7 µM, 1.3% CV, Sample 1 (Total NO Concentration)- 157.8 µM, 1.8% CV Inter Assay Precision: Three samples were further diluted in Assay Buffer and run in duplicates in twenty assays run over multiple days by three operators. The mean and precision of the calculated Nitrite or Total NO concentrations were: Sample 1 (Nitrite Concentration)- 44.1 µM, 3.1% CV, Sample 1 (Total NO Concentration)- 68.8 µM, 7.4% CV Sample 2 (Nitrite Concentration)- 66.4 µM, 4.0% CV, Sample 1 (Total NO Concentration)- 112.1 µM, 5.7% CV Sample 3 (Nitrite Concentration)- 126.7 µM, 6.3% CV, Sample 1 (Total NO Concentration)- 154.48 µM, 4.1% CV
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Assay cross reactivity
Species Independent
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Samples to be used with this kit
Serum, Plasma, Urine, Saliva, Water, Buffer, Cell Lysates, Tissue Culture Media
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Maximum samples to be used with this kit
88 samples in duplicate
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Assay duration
30 Minutes
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Kit s sensitivity
2,63 µM in the Nitrite and 1,02 µM in the Total Nitric Oxide
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Assay detection limit
3,125 - 200 µM
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Storage recommendations
4°C
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Shipping recommendations
Blue Ice
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Bibliography
1. Moncada, S and EA Higgs. (1991) Eur. J. Clin. Invest., 21:361-374. 2. Kapil, V. et al. (2010) Heart, 96:1703-1709. 3. Seth, D and Stamler, JS. (2007) Curr. Opin. Chem. Biol., 15:1-8. 4. Eursalimsky, JD and Moncada, S. (2007) ATVB 27:2524-2531. 5. Knott, AB and Bossy-Wetzel, E. (2010) Diab.Obes.Metab. 12(Suppl2):126-133. 6. Van Dyke, K. et al. (2010) Ann. N.Y. Acad. Sci. 1203:138-145. 7. Reher, VSG. et al. (2007) J. Oral Sci. 49(4):271-276. 8. Sogut, S. et al. (2003) Clin. Chim. Acta 331:111-117. 9. Balam. E. et al. (2002) Jpn. J. Clin. Oncol. 32(5):162-166. 10. Moshage, H. (1997) Clin.Chem. 43(4):553-556.
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Release date
17-Sep-2014
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PubMed number
Refer to PubMed
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Tested applications
No data available / currently testing
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Tested reactivity
No data available / currently testing
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Resources available upon request
Kit Booklet, MSDS
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Product image link
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Product image legend
Typical Standard Curve for Nitric Oxide Detection Kit StressXpress® - SKT-212. Assay Type: Coupled Enzyme Assay. Detection Method: Colorimetric Assay. Assay Range: 3.125 - 200 uM. | Linearity was determined by taking two human urine samples with known Nitrite and Total Nitric Oxide Concentrations and mixing them in given ratios. The measured concentrations were compared to the expected values based on the ratios used. Graph of the Typical Standard Curve for the Nitric Oxide Detection Kit StressXpress - SKT-212 | Graph of the Linearity Recovery for the Nitric Oxide Detection Kit StressXpress - SKT-212
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Warnings
Non-hazardous
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Country of production
Canada
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Total weight kg
2
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Net weight g
300
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Description
The detections of the targets with this kit is a type of test that can be performed on any target containing biological samples after clean up of interfering agents. The assay must be performed following the protocol.
