• Detection method- Fluorescence (Ex/Em 380/461 nm)
• Sample type- Cell and Tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids
• Species reactivity- Mammalian
• Applications- Measurement of total nitrate/nitrite concentration
Features & Benefits:
• Simple procedure; takes only 2-3 hours
• Fast and convenient
• The fluorometric kit provides a simple two-step process for quantitative measurement of total nitrate/nitrite productions. The assay can be easily done in a 96-well plate.
• Assay Buffer
• Enzyme Cofactor
• Nitrate Reductase
• Nitrate Standard
• Nitrite Standard
• DAN Probe
• Sodium Hydroxide
• Microtiter plate
• Plate Cover
Nitric oxide (NO) plays an important role in neurotransmission, vascular regulation, immune response and apoptosis. Since NO is rapidly converted to nitrite (NO²ˉ) and nitrate (NOᶟˉ), the total concentration of nitrite and nitrate is used as a quantitative measure of NO production. BioVision’s Nitric Oxide Fluorometric Assay Kit provides an accurate and convenient measurement of total nitrate/nitrite concentration in a simple two-step process. In the first step nitrate is converted to nitrite by nitrate reductase. In the second step, nitrite reacts with the fluorescent probe DAN (2, 3 diaminonaphthalene). 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.
USAGE: For Research Use Only! Not For Use in Humans.
What anticoagulant works best for plasma prep for this assay?
You can use either heparin or EDTA for plasma prep for this assay. Conventionally EDTA should be avoided when assaying for enzyme analytes. Other than this restriction, you can use either, unless otherwise specified.
What are the main differences between K252-200 and K262-200?
Here is the comparision between these 2 assay kits which can be used for the detection of nitric oxide:
K252 uses fluorometric detection, whereas K262 uses colorimetric detction.
The K252 kit is ~ 10 times more sensitive than the K262 assay kit.
Can we use this kit to measure NO that have been generated during culture period and have been released in the culture medium (RPMI 1640)? I know that NO is not stable and is rapidly converted to nitrite/nitrate!
Yes, this kit can be used with RPMI 1640 culture media for measuring NO. Although the NO is rapidly converted to nitrite and nitrate, this kit will allow the nitrate to also get converted to nitrite and the total nitrite will then react with the probe. So your final reading will be a measure of the total NO released into the media.
Is the Nitrate Reductase provided in this kit NADH dependent or NADPH dependent. What is the sensitivity of this assay kit?
The Nitrate Reductase is NAD(P)H dependent. The detection limit of this assay is 0.03 uM.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kits citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration?
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kits components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, It is recommended to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.