Nitric Oxide Colorimetric Detection Kit

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Antigen
Minimum Detection Limit
3.0 μM
Application
Biochemical Assay (BCA)
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Purpose The DetectX® Nitric Oxide Detection Kit is designed to quantitatively measure Nitrate and Nitritepresent in a variety of samples. Nitric Oxide content is derived from the sum of Nitrate (-NO3) andNitrite (-NO2).
Brand DetectX®
Sample Type Water, Biological Buffers, Serum, Plasma, Urine, Saliva, Tissue Culture Supernatant
Detection Method Colorimetric
Specificity Species Independent. Samples Types validated: Water, Buffers, Serum, Plasma, Urine, Saliva and TCM
Sensitivity 2.63 µM in the Nitrite and 1.02 µM in the Total Nitric Oxide assays.
Characteristics The Nitric Oxide (NO) 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 and Nitrite. 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. 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 in- cubation at room temperature, Color Reagents A and B are added and incubated at room tempera- ture for 5 minutes. The concentration of Nitrate in the sample is calculated by taking the measured Nitrite concentration and subtracting from the Total Nitric Oxide concentration for the sample.
Components Clear 96 well Plates 2 Plates
Nitrate Standard 200 μL Sodium Nitrate at 2,000 μM in a special stabilizing solution. Calibrated to NIST Standard Reference Material Lot Number 3185 Nitrite Standard 200 μL
Sodium Nitrite at 2,000 μM in a special stabilizing solution. Calibrated to ISO/IEC 17025
Assay Buffer 60 mL A buffer containing detergents and stabilizers.
NADH Concentrate 1.2 mL Reduced ß-nicotinamide adenine dinucleotide (NADH) as a stable solution.
Nitrate Reductase 1 Vial Nitrate Reductase (NR) as a stable solid stored in a desiccator.
Enzyme Stabilization Buffer 1 mL A buffer containing special stabilizers for NR.
Color Reagent A 5 mL A solution of Sulfanilamide in acid. CAUTION: CAUSTIC
Color Reagent B 5 mL A solution of N-(1-Naphthyl)ethylenediamine in acid. CAUTION: CAUSTIC
Material not included Distilled or deionized water free of detectable nitrate or nitrite. 10,000 Molecular Weight Cut Off (MWCO) polysulfone filters (Corning Spin-X UF 500, 431478) or similar product.
Repeater pipets using disposable tips for addition of Color Reagents A & B, NADH and Nitrate Reductase. 96 well plate reader capable of reading optical absorption at 540-570 nm.
Software for converting optical density (OD) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
Alternative Name Nitric Oxide
Background 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 systems1. NO is bio-available by two routes. It can be endogenously generated by constitutive or induced en- zymes like Nitric Oxide Synthase or it can be orally ingested as nitrates / nitrites for rapid uptake into circulation and subsequent conversion2. 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 con- verted 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 signaling3. The balance and dynamics of the regulatory/damage facets of NO are major forces in mitochondrial signaling and dysfunction4. NO is linked not only to coronary heart disease, en- dothelial dysfunctions, erectile dysfunction, and neurological disorders, but also diabetes, chronic periodontitis, autism, cancer, and assorted age-related diseases5-9. The physical properties of Nitric Oxide make it challenging for direct detection methods. How- ever, colorimetric methods can be applied to measure its stable break-down products nitrate (-NO ) and nitrite (- 103 NO2)
Application Notes NO, Nitrate and Nitrite is identical across species and this kit will measure NO from all sources.
We determined NO in human samples and the end user should evaluate recoveries of NO in samples from other species being tested.
The kit will measure NO in cell culture medium, however many media contain nitrate salts.
Care needs to be taken in the selection of media when NO measure- ment is to be done.
If samples need to be stored after collection, we recommend storing them at -70 °C or lower, pref- erably after being frozen in liquid nitrogen.
This assay has been validated for serum, plasma, urine, and saliva, as well as water and buffer samples.
Tris, HEPES, and PBS buffers are compatible at pH 7.2, as is EDTA at ≤ 10 mM.
Detergents such as Triton X-100, Tween 20 and CHAPS are compatible at concentrations of ≤ 0.1 % .
Most cell lysates and tissue homogenates should also be compatible.
Samples containing these detergents should be diluted at least 1:2 with the Assay Buffer.
Samples containing SDS or azide are not compatible with the assay.
Samples containing visible particu- late should be centrifuged prior to filtration and using.
Assay Time 0.5 h
Plate 96 wells
Protocol 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 tem- perature for 5 minutes.
The colored product is read and calculated as with the Nitrite determina- tion 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.
This kit uses Nitrate and Nitrite Standard solutions calibrated to the US National Institute for Sci- ence and Technology Standard Reference Materials and ISO/IEC standards.
Reagent Preparation

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine NO con- centrations.
Ensure that all samples have reached room temperature and have been diluted and filtered through a 10,000 MWCO filter prior to running them in the kit.
Nitrate Reductase (NR) Allow the desiccator to warm to room temperature.
Add 550 μL of Enzyme Stabilization Buffer to the vial.
Vortex gently and allow to sit at room temperature for 5 minutes.
For extended periods of time (>2 hours) store reconstituted NR on ice.
Store any unused reconstituted NR at -20 °C.
Prepare NR for use in the assay by taking one part of reconstituted NR and adding to three parts of Assay Buffer.
See Table below.
Nitrate Reductase Dilution Table 1/2 Plate One Plate Two Plates Reconstituted NR 150 μL 275 μL 500 μL Assay Buffer 450 μL 825 μL 1.5 mL Total Volume 600 μL 1.1 mL 2 mL ® For extended periods of time (>2 hours) store reconstituted NR on ice. www.ArborAssays.com 7 WEB INSERT 150513 reagent preparatiOn cOntinued NADH Preparation Prepare NADH by diluting one part of NADH Concentrate with an equal part of Assay Buffer.
NADH Dilution Table 1/2 Plate One Plate Two Plates NADH Concentrate 300 μL 550 μL 1 mL Assay Buffer 300 μL 550 μL 1 mL Total Reaction Mix Volume 600 μL 1.1 mL 2 mL Do not store diluted NADH.
Standard Preparation Nitrate and Nitrite Standards are prepared identically by labeling seven test tubes as #1 through #7.
Briefly vortex to mix and then spin the vial of standard in a microcentrifuge to ensure contents are at bottom of vial.
Pipet 360 μL of Assay Buffer into tube #1 and 200 μL into tubes #2 to #7.
Care- fully add 40 μL of either the -NO2 or -NO3 Standard to tube #1 and vortex completely.
Take 200 μL of the solution in tube #1 and add it to tube #2 and vortex completely.
Repeat this for tubes #3 through #7.
The concentration of Nitrate or Nitrite in tubes 1 through 7 will be 200, 100, 50, 25, 12.5, 6.25 and 3.125 μM.
Use all Standards within 2 hours of preparation.

Sample Preparation

All samples must be filtered through a 10,000 MWCO spin filter to remove protein. Serum, plasma, saliva, or urine Dilute sample with Assay Buffer and filter through a 10,000 MWCO device following the manu- facturer's recommendations. Collect the filtrates and either further dilute with Assay Buffer as appropriate or use directly in the assay. For serum and plasma, the recommended final dilution is ≥1:4. For urine and saliva, the recommended final dilution is ≥1:8.

Assay Procedure

Use the appropriate standards for either Nitrite (-NO2) or Nitrate ( -NO3) determination. All sam- ples should be diluted and filtered through a 10,000 MWCO filter prior to using. Nitrite Determination Protocol
1. Use the plate layout sheet on the back page to aid in proper sample and standard identification.
2. Pipet 50 μL of samples or Nitrite standards into duplicate wells in the plate.
3. Pipet 50 μL of Assay Buffer into duplicate wells as the Zero standard.
4. Add 25 μL of the Color Reagent A to each well using a repeater pipet.
5. Add 25 μL of the Color Reagent B to each of well using a repeater pipet.
6. Incubate at room temperature for 5 minutes.
7. Read the optical density at 540-570 nm. These readings are for the Nitrite determination. Total Nitric Oxide Determination Protocol
1. Use the plate layout sheet on the back page to aid in proper sample and standard identification.
2. Pipet 50 μL of samples or Nitrate standards into duplicate wells in the plate.
3. Pipet 50 μL of Assay Buffer into duplicate wells as the Zero standard.
4. Add 10 μL of prepared NADH to each well using a repeater pipet.
5. Add 10 μL of prepared NR to each well using a repeater pipet.
6. Incubate at room temperature for 20 minutes.
7. Add 25 μL of the Color Reagent A to each well using a repeater pipet.
8. Add 25 μL of the Color Reagent B to each of well using a repeater pipet.
9. Incubate at room temperature for 5 minutes. 10. Read the optical density at 540-570 nm. These readings are for the Total Nitric Oxide determination.

Calculation of Results

Average the duplicate optical density readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean ODs for the zero standard.
The concentrations obtained should be multiplied by the dilution factor to obtain sample values.
Or, use the online tool from http://www.myassays.com/arbor-assays-nitric-oxide-colorimetric-kit. assay to calculate the data.
QR code for Data Analysis: *The MyAssays logo is a registered trademark of MyAssays Ltd.
Nitrite (-NO2) concentrations are calculated from the data obtained from the Nitrite Protocol stan- dard curve data utilizing the curve fitting routine supplied with the plate reader.
Total NO concentrations are calculated from the data obtained from the Total Nitric Oxide Proto- col (nitrite + nitrate) standard curve data utilizing the curve fitting routine supplied with the plate reader.
Nitrate (-NO3) concentrations are obtained by subtracting the -NO2 concentrations of each sample from the Total NO concentrations.
See Below: Nitrate (-NO3) = Total NO - Nitrite ( -NO2)

Restrictions For Research Use only
Precaution of Use As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The Color Reagents A and B are both acid solutions and should be handled like any laboratory acid.
Storage -20 °C,4 °C,RT
Storage Comment All components of this kit should be stored at 4°C until the expiration date of the kit. Once reconstituted, the Nitrate Reductase must be stored at -20°C.
Supplier Images
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Adela, Nethi, Bagul, Barui, Mattapally, Kuncha, Patra, Reddy, Banerjee: "Hyperglycaemia enhances nitric oxide production in diabetes: a study from South Indian patients." in: PLoS ONE, Vol. 10, Issue 4, pp. e0125270, 2015 (PubMed).

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Sawicka, Długosz, Rembacz, Guzik: "The effects of coenzyme Q10 and baicalin in cisplatin-induced lipid peroxidation and nitrosative stress." in: Acta poloniae pharmaceutica, Vol. 70, Issue 6, pp. 977-85, 2014 (PubMed).