Nitric Oxide (NO) is a gaseous free radical with a short half-life in vivo of a few seconds or less. It is a pleiotropic biological mediator that regulates diverse activities ranging from neuronal function to immune system regulation. NO is catalyzed by enzymes of the Nitric Oxide Synthase (NOS) family, which include neuronal NOS (nNOS/NOS1), inducible NOS (iNOS/NOS2), and endothelial NOS (eNOS/NOS3). NO is lipid soluble, so it is not stored by synthesized de novo and freely diffuses across lipid membranes. NO mediates its effects on target cells via several different mechanisms. For instance, NO can activate Guanylyl Cyclase, which catalyzes the formation of the second messenger cGMP. cGMP is implicated with a range of biological functions such as regulating smooth muscle contractility, cell survival, proliferation, axon guidance, synaptic plasticity, inflammation, angiogenesis, and the activity of cyclic nucleotide-gated channels. NO also functions as an anti-tumor and anti-microbial agent via mechanisms that include its conversion to peroxynitrite, the formation of S-nitrosothiols, and the depletion of arginine. Additionally, NO suppresses mitochondrial respiration through the inhibition of Cytochrome Oxidase, and may modify protein activity through post-translational nitrosylation. Altered levels of NO have been shown to be associated with sepsis, reproduction, infection, hypertension, exercise, type 2 diabetes, hypoxia, and cancer.
Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit
R&D Systems | Catalog # KGE001
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Key Product Details
Assay Length
1 hour
Sample Type & Volume Required Per Well
Cell Culture Supernates (10 µL), Serum (25 µL), EDTA Plasma (25 µL), Heparin Plasma (25 µL), Citrate Plasma (25 µL), Urine (10 µL)
Sensitivity
0.78 µmol/L
Assay Range
3.12-200 µmol/L (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma, Citrate Plasma, Urine)
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Product Summary for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit
The Parameter Total Nitric Oxide kit has two assay options. Endogenous nitrite is measured in the first option. In the second option, nitrate is converted to nitrite using nitrate reductase and total nitrite is measured. To obtain the nitrate concentration, endogenous nitrite is subtracted from the total nitrite value.
Product Specifications
Format
96-well strip plate
Measurement
Quantitative ELISA
Detection Method
Colorimetric - 450nm (TMB)
Species
Multi-Species
Specificity
Measures Total Nitric Oxide, Nitrite and Nitrate levels in various samples
Cross-reactivity
< 0.5% cross-reactivity observed with available related molecules. < 50% cross-species reactivity observed with species tested.
Interference
Interference observed with other substances.
Precision
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
Cell Culture Supernates, Citrate Plasma, EDTA Plasma, Heparin Plasma, Serum, Urine
| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 40 | 40 | 40 |
| Mean (µmol/L) | 30.0 | 77.2 | 138.0 | 31.5 | 79.9 | 138.7 |
| Standard Deviation | 0.76 | 1.10 | 2.20 | 1.46 | 2.91 | 4.78 |
| CV% | 2.5 | 1.4 | 1.6 | 4.6 | 3.6 | 3.4 |
Recovery for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit
The recovery of the nitrate standard spiked to levels throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average % Recovery | Range % |
|---|---|---|
| Cell Culture Supernates (Nitrite Assay) (n=10) | 107 | 91-115 |
| Plasma (Nitrate Assay) (n=25) | 98 | 87-110 |
| Plasma (Nitrite Assay) (n=20) | 104 | 92-117 |
| Serum (Nitrate Assay) (n=10) | 95 | 89-101 |
| Serum (Nitrite Assay) (n=10) | 109 | 98-118 |
| Urine (Nitrite Assay) (n=10) | 101 | 87-112 |
Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of the nitrate standard were serially diluted with the Reaction Diluent to produce samples with values within the dynamic range of the assay.
Scientific Data Images for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit
Multi-species Total Nitric Oxide and Nitrate/Nitrite ELISA Standard Curve
Preparation and Storage
Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: Nitric Oxide
Additional Nitric Oxide Products
Product Documents for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit
For research use only
Citations for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit
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Customer Reviews for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit (7)
3.9 out of 5
7 Customer Ratings
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Sample Tested: Cell Culture Media and IPS2 induced pluripotent stem cellsVerified Customer | Posted 09/03/2025It is good products if not considering the cost.
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Sample Tested: Cell culture supernatantVerified Customer | Posted 09/18/2022
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Sample Tested: Cell culture supernatantVerified Customer | Posted 10/19/2020
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Sample Tested: THP-1 human acute monocytic leukemia cell lineVerified Customer | Posted 03/12/2019
Bio-Techne ResponseThank you for reviewing our product. We are sorry to hear that this product did not perform as expected. We have been in touch with the customer to resolve this issue according to our Product Guarantee and to the customer’s satisfaction.
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Sample Tested: Cell culture supernatantVerified Customer | Posted 02/26/2019
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Sample Tested: Cell Culture MediaVerified Customer | Posted 04/02/2018
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Sample Tested: Human serumVerified Customer | Posted 08/23/2016I did spike in study for both nitrite and nitrate assay. The recovery of Nitrite is very good. However the nitrate is very poor.
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Protocols
View specific protocols for Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit (KGE001):
Nitrite Assay Procedure
Nitrate Reduction Assay Procedure
Refer to the product for complete assay procedure.
Refer to the product for complete assay procedure.
Nitrate Reduction Assay Procedure
Refer to the product
This assay procedure measures the concentration of endogenous nitrite present in the sample. Bring all reagents and samples to room temperature before use. It is recommended that all samples and standards be assayed in duplicate.
- Prepare all reagents, standard dilutions, and samples as directed in the product insert.
- Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
- Add 50 µL of Reaction Diluent (1X) to the Blank wells.
- Add 50 µL of Nitrite Standard or sample to the remaining wells.
- Add 50 µL of Reaction Diluent (1X) to all wells.
- Add 50 µL of Griess Reagent I to all wells.
- Add 50 µL of Griess Reagent II to all wells. Mix by gently tapping the side of the plate.
- Incubate at room temperature for 10 minutes. Read at 540 nm with wavelength correction at 690 nm.
50 µL Reaction Diluent (1X)
50 µL Nitrite Standard or Sample
50 µL Reaction Diluent (1X)
50 µL Griess Reagent I
50 µL Griess Reagent II
Refer to the product
This assay procedure measures total nitrite by converting nitrate to nitrite. To determine the nitrate concentration in the sample, the endogenous nitrite concentration measured from the Nitrite Assay Procedure must be subtracted from the converted nitrite concentration measured in this procedure. Bring all reagents and samples to room temperature before use. It is recommended that all samples and standards be assayed in duplicate.
- Prepare all reagents, standard dilutions, and samples as directed in the product insert.
- Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
- Add 50 µL of Reaction Diluent (1X) to the Blank wells.
- Add 50 µL of Nitrate Standard or sample to the remaining wells.
- Add 25 µL of NADH to all wells.
- Add 25 µL of diluted Nitrate Reductase to all wells. Mix well, cover with a plate sealer, and incubate at 37 °C for 30 minutes.
- Add 50 µL of Griess Reagent I to all wells.
- Add 50 µL of Griess Reagent II to all wells. Mix by gently tapping the side of the plate.
- Incubate at room temperature for 10 minutes. Read at 540 nm with wavelength correction at 690 nm.
50 µL Reaction Diluent (1X)
50 µL Nitrate Standard or Sample
25 µL NADH
25 µL Nitrate Reductase
50 µL Griess Reagent I
50 µL Griess Reagent II
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