Nitric Oxide Assay Kit; Fluorometric

Nitric Oxide Assay Kit; Fluorometric

Stock Code: 3586512
Manufacturer Part No: 482655-1KIT
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Components


Assay Buffer, Nitrate Reductase, Enzyme Cofactors, Nitrate Standard, Nitrite Standard, DAN Reagent, NaOH, Microtiter Plates, Plate Covers, and a user protocol.


General description


Assay kit useful for the rapid quantitative measurement of nitric oxide (NO). Displays 50-fold increased sensitivity over the colorimetric nitric oxide assay kit (Cat. No. 482650). The assay is based on the enzymatic conversion of nitrate to nitrite by nitrate reductase, followed by the addition of 2,3-diaminonapthalene (DAN), and NaOH, which converts nitrite to a fluorescent compound. Fluorescence measurements of this compound accurately determine the nitrite (NO2-) concentration (excitation max.: 365 nm; emission max.: 450 nm).Do not use with nitrate- or nitrite-containing tissue culture media such as RPMI.


Legal Information


CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany


Other Notes


Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.


Miles, A.M., et al. 1996. Methods Enzymol. 268, 105.Misko, T.P., et al. 1993. Anal. Biochem. 214, 11.Green, L.C., et al. 1982. Anal. Biochem.126, 131.


Packaging


1 kit in Fibre case


Preparation Note


1. Culture Medium: Culture medium such as RPMI 1640 may contain high levels of nitrate. It is best not to use these types of media, particularly when small changes in nitrate levels are measured. If it is absolutely necessary to use this type of medium then cellular nitrate/nitrite levels can be quantitated by subtracting the level of nitrate/nitrite in the medium (in the absence of cells) from the total levels. Phenol red and fetal bovine serum (FBS) added to the medium can cause a significant reduction in fluorescence. Whenever possible these components should be avoided. The effect of media components on fluorescence intensity must be assessed by making the nitrate or nitrite standard curve in the presence of an equivalent amount of the phenol red or FBS. To obtain maximum signal response, it is best to use 10 or 20 µl sample volumes. Use of larger sample volumes (30 to 50% of the final reaction volume) can lead to quenching of fluorescence. To prepare a standard curve in the presence of media, simply prepare the nitrate or nitrite standard curve substituting the amount of media desired in the place of assay buffer. For the measurement of nitrate plus nitrite an incubation period of 1 h is required for the reaction to reach completion.2. Plasma or Serum: Ultrafilter plasma or serum samples through a 10 or 30 kDa cut-off filter using a commercially available centrifuge or microfuge ultrafiltration device. This procedure removes hemoglobin thereby avoiding the reduction in fluorescence intensity. Assay for nitrate and/or nitrite using a maximum of 10 µl filtrate. The conversion of nitrate to nitrite requires 1-2 h (for ≥95% conversion).3. Tissue Homogenates:

Quality Level100
ManufacturerSIGMA-ALDRICH
Storage Temp.−20°C

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