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Nitric Oxide Assay Kit (Colorimetric) (ab65328)

Reviews (2)Q&A (27)References (95)
Functional Studies - Nitric Oxide Assay Kit (Colorimetric) (ab65328)
  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)
  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)
  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)
  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 1 hr 30 min
  • Sample type: Cell Lysate, Other biological fluids, Plasma, Serum, Tissue Extracts, Urine
  • Sensitivity: 1 nmol/well

Overview

  • Product name

    Nitric Oxide Assay Kit (Colorimetric)
    See all Nitric oxide kits

  • Detection method

    Colorimetric

  • Sample type

    Urine, Serum, Plasma, Other biological fluids, Tissue Extracts, Cell Lysate

  • Assay type

    Quantitative

  • Sensitivity

    1 nmol/well

  • Range

    1 nmol/well - 10 nmol/well

  • Assay time

    1h 30m

  • Species reactivity

    Reacts with: Mammals, Other species

  • Product overview

    Nitric Oxide Assay Kit (Colorimetric) ab65328 provides an accurate, convenient measure of total nitrate/nitrite in a simple two-step process.


    In the nitric oxide assay protocol:
    - the first step converts nitrate to nitrite by using nitrate reductase
    - the second step uses Griess Reagent to convert nitrite to a deep purple azo compound.
    The amount of the azochromophore accurately reflects nitric oxide amount in samples.


    Nitric oxide assay protocol summary:
    - add samples and standards to wells
    - add nitrate reductase and enzyme cofactor and incubate for 1 hr at room temp to convert nitrate to nitrite
    - add enhancer and incubate for 10 min at room temp
    - add Griess Reagent R1 and Griess Reagent R2
    - analyze with microplate reader

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K262 Nitric Oxide Colorimetric Assay Kit. K262-200 is the same size as the 2 96 tests size of ab65328.

    If you are interested in a fluorometric detection kit, please check Nitric Oxide Assay Kit (Fluorometric) (ab65327).

    For an assay kit to detect nitrite alone see Griess Reagent Kit ab234044.

    Nitric oxide (NO) plays an important role in neurotransmission, vascular regulation, immune response and apoptosis. NO is rapidly oxidized to nitrite and nitrate which are used to quantitate NO production.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 2 x 96 tests
    Assay Buffer WM 1 x 30ml
    Enhancer (Lyophilized) Purple 1 vial
    Enzyme Cofactor (Lyophilized) Blue 1 vial
    Griess Reagent R1 NM 1 x 10ml
    Griess Reagent R2 Amber NM 1 x 10ml
    Microtiter plate 2 units
    Nitrate Reductase (Lyophilized) Green 1 vial
    Nitrate Standard (Lyophilized) Yellow 1 vial
    Nitrite Standard (Lyophilized) Orange 1 vial
    Plate Cover 2 units

  • Research areas

  • Relevance

    Nitric oxide (NO) is a key vertebrate biological messenger, playing an important role in neurotransmission, vascular regulation, immune responses and apoptosis. NO , also known as "endothelium-derived relaxing factor" or "EDRF", is synthesized from L-arginine, oxygen and NADPH by various NO synthases. Most of the NO in the cell is oxidized to nitrite (NO2-) and nitrate (NO3-), and therefore the concentrations of these anions are generally as a quantitative measure of NO production.

Images

  • Functional Studies - Nitric Oxide Assay Kit (Colorimetric) (ab65328)
    Functional Studies - Nitric Oxide Assay Kit (Colorimetric) (ab65328)Nam, Eunryel et al., Scientific reports vol. 8,1 7312., Fig 4 , doi:10.1038/s41598-018-25786-x
    Blood NOx concentration measured with ab65328. Blood NOx concentration after application of the NE with NO. The level of blood NOx concentration increased 3-fold after the application of the NE with NO compared with control group.
  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)
    Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)

    Nitrite and nitrate measured in cell lysates showing quantity (micromol) per 106 cells

  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)
    Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)

    Nitrite and nitrate measured in biologicals showing concentration (micromolar)

  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)
    Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)

    Standard curve (colorimetric) : mean of duplicates (+/-SD) with background readings subtracted

  • Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)
    Functional Studies - Nitric Oxide Colorimetric Assay Kit (ab65328)

    Standard curve (Colorimetric) : mean of duplicates (+/-SD) with background readings subtracted

References (95)

Publishing research using ab65328? Please let us know so that we can cite the reference in this datasheet.

ab65328 has been referenced in 95 publications.

  • Feng Y  et al. Pyruvate kinase M2 (PKM2) improve symptoms of post-ischemic stroke depression by activating VEGF to mediate the MAPK/ERK pathway. Brain Behav 12:e2450 (2022). PubMed: 34898024
  • Lin YK  et al. Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory a7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm. Oxid Med Cell Longev 2022:9964689 (2022). PubMed: 35096275
  • Abd-Elhakim YM  et al. Time-dependent expression of high-mobility group box-1 and toll-like receptors proteins as potential determinants of skin wound age in rats: Forensic implication. Int J Legal Med 136:1781-1789 (2022). PubMed: 35132471
  • Lau M  et al. Enhanced Antioxidant Effects of the Anti-Inflammatory Compound Probucol When Released from Mesoporous Silica Particles. Pharmaceutics 14:N/A (2022). PubMed: 35335878
  • Mangmool S  et al. Anti-Inflammatory Effects of Mitrephora sirikitiae Leaf Extract and Isolated Lignans in RAW 264.7 Cells. Molecules 27:N/A (2022). PubMed: 35630789
View all Publications for this product

Customer reviews and Q&As

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1-10 of 29 Abreviews or Q&A

Rat Plasma

 Good Below average 2/5 (Ease of Use)
Abreviews
abreview image
Overall the test worked well for us. All of the required information was included, but the way that the steps were structured was awkward at times. This made the protocol difficult to follow.
Specifically, the section describing how to deproteinize your sample (step 11.4.4 in protocol booklet). An online protocol is also available from abcam, and was a little more clear (https://www.abcam.com/protocols/deproteinization-protocol). It would be nice if these protocols were updated to be more clear and uniform across abcam.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Feb 27 2018

Use of nitric oxide colormetric assay on avian plasma samples

 Average Good 4/5 (Ease of Use)
Abreviews
I used this kit to measure nitric oxide (nitrate and nitrite combined) in the plasma of adult male red-winged blackbirds (Agelaius phoeniceus) housed in an outdoor aviary. Preliminary data suggests that the assay requires a minimum of 15uL of plasma per well (ie, 30uL plasma to run in duplicate) to produce repeatable results. Thus far, we have found birds to have levels of NOx between 14-250 uM. It appears that birds with an active infection could have elevated NOx.
Preliminary data also suggests that error can be easily introduced when ready-ing the samples for the assay by deproteinating plasma with PCA. I suspect that this is particularly problematic for samples with absorbance reading less than 0.2, but we need more evidence to be certain.
I did notice that the instructions on the web site were not the same as in the booklet sent. In the booklet I received, the instructions said to use the plate reader after adding the second Griess reagent with no wait time listed. On the online book an incubation time was listed (10 min). There were also inconsistencies in the the list of reagents provided and what was actually provided (We got plates, which is great!). The plates were labeled with a store in -20C, even though they're just plastic plates that isn't necessary.
Technical support was very helpful in answering questions quickly whenever I called - was really happy with the support.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Feb 01 2016

Question

Inquiry: My assay buffer got spilled what can I use to replace it? Is PBS ok?

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Answer



I am happy to confirm that extra assay buffer can now be purchased using the following product number:

ab171368

https://www.abcam.com/Nitric-Oxide-Assay-Buffer-ab171368.html (or use the following: https://www.abcam.com/Nitric-Oxide-Assay-Buffer-ab171368.html).

PBS is not a suitable replacement for the assay buffer.

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Question

appel- est-ce que les échantillons (surnagéants de culture)peuvent être congélées avant l'experience?

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Answer

Merci pour votre patience.

Le laboratoire vient de me confirmer que le NO est stable et que les échantillons de surnagéant aussi peuvent donc être conservées en aliquots à-20°C ou -80°C.

J'espère que cet information vous est utile. N'hésitez pas à nous recontacter si vous avez des autres questions.

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Question

which one of these 2 kits is more sensitive?

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Answer

Thank you for contacting us. The Fluorometric assay kit ab65327 is much more sensitive (about 10x) than kit ab65328 (colorimetric assay kit). For ab65328 (colorimetric): The absorbance of samples should be in the linear range of the standard curve (0-10 nmol/well). The detection limit of the assay is approximately 0.1 nmol nitrite/well, or 1 µM. For ab65327 (fluorometric): The fluorescence of samples should be in the linear range of the standard curve (0-1000 pmol/well). A 1000 pmol = 1 nmol. The detection limit of the assay is approximately 10 pmol nitrite/well, or 0.1 µM. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

Can this kit be used with MEM tissue culture medium that contains phenol red?

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Answer

Thank you for your enquiry. This kit will be compatible with MEM with phenol red. Media that contain phenol red as a pH indicator do not interfere with the Griess reaction as the indicator is typically yellow colored under the conditions of the Griess reaction. You want to be sure to avoid media that contain nitrates, such as RPMI which contains calcium nitrate. MEM does not. Also, NO synthetase enzymes utilize high concentrations (0.5-1 mM) of NADPH which may inhibit the Griess color reaction slightly. If your know that your samples contain NO synthetase, you will want to take this into consideration when interpreting your data. I hope this information helps. Please do not hesitate to contact us if you need any more advice or information.

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Question

after you add the enhancer and incubate for 10 mins you then add grey as rehang r1 and then r2. Do you wait and incubate after adding the greiss regent r1? And how long after addition of the regent r2 do you need to wait before you can you measure outputs?

Read More
Answer

fter adding Griess Reagent R2 in standard and unknown leave it for 10 minutes at room temperature. The color developed will be stable for an hour.   

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Question

Is heparin lithium is OK for treating plasma samples used with these 2 kits?

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Answer

Yes, heparin should be fine to use with ab65328 and ab118970.

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Question

Our lysis buffer has salts, triton X-100 and protease inhibitors. Do you have any information about which components would interfere with the NO analysis

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Answer



Standard amounts of salts, Triton and protease inhibitors should be alright. We have not tested this kit’s function using high salt/detergent but theoretically small amounts of these should not interfere with the assay.

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Question

I was wondering if you could let me know if the product above includes a step to remove proteins? Or if it needs one?

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Answer


This kit does not include a step to remove proteins. Depending on your sample type, it is however recommended to use 10kD spin columns (ab93349) to deproteinate your samples.

For example, it is recommended for serum samples since there are proteins in the serum which can interfere with the nitrite level but this may not be necessary for cell lysates.

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1-10 of 29 Abreviews or Q&A

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