Overview

  • Product name

    Nitric Oxide Assay Kit (Fluorometric)
    See all Nitric oxide kits
  • Detection method

    Fluorescent
  • Sample type

    Urine, Serum, Plasma, Other biological fluids, Tissue Extracts, Cell Lysate
  • Assay type

    Quantitative
  • Assay time

    3h 00m
  • Product overview

    Nitric Oxide Assay Kit (Fluorometric) ab65327 provides an accurate, convenient measure of total nitrate/nitrite concentration in a simple two-step process.


    In the nitric oxide assay protocol:
    - the first step converts nitrate to nitrite by nitrate reductase
    - in the second step, nitrite reacts with the fluorescent probe DAN (2, 3 diaminonaphthalene)


    The fluorescence can be measured at Ex/Em = 360/450 nm. NaOH enhances the fluorescent yield. The fluorescent intensity is proportional to the total nitric oxide production. The kit has been tested with culture media, plasma, and tissue homogenates.


    Nitric oxide assay protocol summary:
    - add samples and standards to wells
    - add enzyme cofactor, to measure nitrite only add assay buffer or to measure total nitrate + nitrite add nitrate reductase 
    - incubate for 1-4 hr at room temp to convert nitrate to nitrite
    - add enhancer and incubate for 30 min at room temp
    - add DAN probe and incubate for 10 min at room temp
    - add NaOH and incubate for 10 min at room temp
    - analyze with microplate reader

  • Notes

    If you are interested in a colorimetric detection kit, please check Nitric Oxide Assay Kit (Colorimetric) (ab65328).

    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. Since NO is rapidly converted to nitrite (NO2-) and nitrate (NO3-), the total concentration of nitrite and nitrate is used as a quantitative measure of NO production.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 200 tests
    Assay Buffer WM 1 x 40ml
    DAN Probe Amber Red 1 x 1ml
    Enhancer (Lyophilized) Purple 1 vial
    Enzyme Cofactor (Lyophilized) Blue 1 vial
    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
    Sodium Hydroxide Clear 1 x 1ml
  • 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

  • Effect of arginase inhibitor and L-arginine on eATP-induced reduction of nitric oxide production in HSV endothelial cells.
    HSVEC were either untreated (Ctrl), treated with ATP (2 mM), L-NAME, (100 µM), ATP with L-arginine (L-Arg, 2 mM), or ATP with NOHA (10 µM), for 2 hours. The cells were then stimulated with carbachol (CCH, 1 µM) for 10 minutes and the nitric oxide generated was measured as nitrite using the NO assay kit (ab65327) and relative percent of NO generated was calculated. NO generated with CCH was set as 100%, n = 6 passages, ∗p < 0.05, (One way ANOVA).
  • Nitric oxide measured in cell lysates showing quantity (micromol) per 106 cells tested

  • Nitric oxide measured in biologicals (average of 1:6 and 1:12 dilutions) showing concentration (micromolar)

  • Nitrite, nitrate assay in the presence and absence of nitrate reductase. Assays were performed according to the kit protocol with 1 hour conversion of nitrate to nitrite at Step 5.

Protocols

References

This product has been referenced in:

  • Fodelianaki G  et al. Nerve Growth Factor modulates LPS - induced microglial glycolysis and inflammatory responses. Exp Cell Res 377:10-16 (2019). Read more (PubMed: 30817930) »
  • Murugavel S  et al. Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching. Front Pharmacol 9:737 (2018). Read more (PubMed: 30050438) »
See all 5 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Answer

Thank you for contacting us.

I heard back from the lab and they will check the assay buffer, but they would need to know the lot number you have. If you please could send me your order or PO number (or lot number on the kit), I will let them know.

Also, please send me your data - if possible - so we can take a look.

But as I said on the phone, I'd be happy to replace the kit for you as soon as I have the order number.

I look forward to hear back and assist you further.

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Question
Answer



Me confirman mis compañeros que la enzima Nitrato Reductase contenida en el kit proviene de Aspergillus, y en caso de interesaros podríais comprarla de forma independiente del kit.

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Answer


The lab has confirmd that your lysis buffer would be compatible, and that pretty much any generic lysis buffer with a mild detergent will work.

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Answer

Thank you for contacting us.

For tissue samples: Start with 10-50 mg of the tissue, add 500-1000 μl (or ˜3-4 volumes of tissue size) of the assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Load the supernatant unto a 10kda spin column for deproteinization. Use the elute for your subsequent assays. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.

Indeed, some substances can interfere with the assays and should be avoided in sample preparation. Please see individual kit datasheets for more specific information on which substances may affect the specific kits, but in general, substances such as EDTA (> 0.5mM), ascorbic acid (> 0.2%), sodium azide (> 0.2%), NP-40 and Tween-20 (>1%) are known to interfere with these assays and should be avoided in sample preparation.

I have also attached the paper we reference on our datasheet so that you may see how they prepared their tissues.

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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Answer

Thank you for your enquiry regarding ab65327.

We have nottested this kit in frozen tissue in our lab. However, if the samples were frozen appropriately, there is still a possibility that this kit might work on lysates from such tissues.

Though,the protocol has to be optimized for such use. Here are a few references for this kit you may wish to take a look at:

- Kim, H. et al. Long-term Blockade of Vascular Endothelial Growth Factor Receptor-2 Aggravates the Diabetic Renal Dysfunction Associated with Inactivation of the Akt/eNOS-NO Axis. Nephrol. Dial. Transplant., 2010; 10.1093/ndt/gfq610.

- Leicht, S. F. et. al. Adiponectin Pretreatment Counteracts the Detrimental Effect of a Diabetic Environment on Endothelial Progenitors. Diabetes, Feb 2011; 60: 652 - 661.

- Takeda N. et. al. Differential activation and antagonistic function of HIF- isoforms in macrophages are essential for NO homeostasis Genes & Dev., Mar 2010; 24: 491 - 501.

Hope this information has been helpful for you.

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Answer

Thank you for your reply.
I am sorry that you are not happy with this kit. Please see below for the refund information.

The deproteinization step would apply for the samples, not for the standard curve.

The lab also send me the following comments that you might want to consider:

"Yes, the standards can be made in the media, but that would decrease the reading, not increase it. "Phenol red and serum in cell culture media may decrease the reading, and thus the standard curve should be made in the same media."

If the high RFU are coming from the media, then there seems to be a problem with media and the customer would need to change that. It does not reflect the quality of the kit one way or the other. If the customer were to make the standard without adding any media, she would get a good curve. Yes, this kit is definitely suitable with culture media. They probably need to make sure that their media itself is not contaminated.

Our recentre-QC was done without the media addition for the standards."

I hope this additional information is nevertheless helpful to you.


REFUND Information:

Your credit note ID is xxx.

I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

The credit note ID is for your reference only and does not automatically guarantee the credit.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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Answer

Thank you for your email.

As I had mentioned in a previous email, I would be happy to replace the kit for you. Can you please confirm thatthis is what you prefer or rather a credit or refund?

On the same hand, it is important to us to understand where the issue might come from and to understand the problem to prevent it in the future. Thus, weusually ask customers to provide details of their experiment - which you did and we are grateful for that.

The lab just finished testing the kit at their end and it did work fine. However, I do not know what samples theyused in this re-test.

The lab is not sure either why you get such high signal from addingculture medium. They suggested that thedeproteinization step wouldnot be necessary for the standard curve. What readout to you get when using fresh medium? This is probably what you could use for the standard curve - if you didn't try this already.

I'll arrange the replacement as soon as I hear back from you. I'm sorry about the inconvenience and like to resolve this issue for you as soon as possible.

I look forward to hear back from you.

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Question
Answer

At the moment, we do not know if there is something wrong with the kit (as the lab tested the assay buffer and it was fine) or with the way the samples were prepared or measured.

Can you please let me know what exactly was added to the wells with 0 pmol/well?
Was it only assay buffer or also your sample (deproteinized and diluted DMEMand EGM2).

If you only add assay buffer (and nothing else) into a well what would be the readout?

The standard curve would be prepared without any DMEM or EGM2 in a well.

Please let me know soon so that we can find the cause of the high readouts you are getting.

Yes, I'd be happy to send you a replacement, but it is also important for us tounderstand where the cause of the problem might lie.
We currently only have the same lot in stock. I can send you another kit of the same lot, if that's what you prefer. Please let me know and I will arrange for it.

I look forward to hear back from you.

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Answer

Thank you for your email.
I will pass the data and information to the lab for evaluation.

In the meantime, they have checked the assay buffer andit did not produce any RFU in their QC testing done this morning. Thus, the buffer seems to be fine on their end.

I will let you know if the lab has any further suggestions regarding your data. I will be in touch.

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Answer

Thank you for the information. I found your order in the system.


Could you please clarify what Group A, B, and C stand for or maybe how they differ? Are these the same samples in triplicate?
Column 1 thru 6 contain data, but what is the concentration range (i.e. which data would go onto the x-axis if you would plot the data)?

For the standard curve, do you have the fluorescence readout (y-axis) and the concentration (x-axis)? If I understand your data correctly these seem to refer to your samples EGM2 and DMEM, right?

I will get in touch with thelab, but please send me the requested information about your data file.This will be of great help. Thank you so much!

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1-10 of 18 Abreviews or Q&A

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