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482650 Nitric Oxide Assay Kit, Colorimetric

482650
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Detection Methods
Colorimetric

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      Description
      OverviewAssay kit for the rapid quantitative measurement of total nitric oxide (NO). Based on the enzymatic conversion of nitrate to nitrite by nitrate reductase, followed by the spectrophotometric quantitation of nitrite levels using Griess Reagent. Do not use with nitrate- or nitrite-containing tissue culture media such as RPMI.
      Catalogue Number482650
      Brand Family Calbiochem®
      Materials Required but Not Delivered Distilled or deionized water
      Microplate reader with 540 nm filter. NOTE: The wavelength of the filter can be 530 to 560 nm, however, maximum absorbance occurs at 540 nm.
      Precision pipettes ranging from 1 µl to 100 µl, and disposable pipette tips. NOTE: If all 96 wells are to be used at one time, it is suggested that a multichannel pipettor be used.
      Clean test tubes
      Vortex mixer
      References
      ReferencesNims, R.W., et al. 1996. Methods Enzymol. 268, 93.
      Product Information
      Detection methodColorimetric
      Form80 Tests
      Format96-well plate
      Kit containsRecombinant Zea mays Nitrate Reductase, NADH, Nitrate Standard, Color Reagents, Buffer, 96-Well Plate, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time1-1.5 h
      Sample TypeAqueous solutions
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 34-36/37/38-40-42/43-20/21/22

      Causes burns.
      Irritating to eyes, respiratory system and skin.
      Limited evidence of a carcinogenic effect.
      May cause sensitization by inhalation and skin contact.
      Harmful by inhalation, in contact with skin and if swallowed.
      S PhraseS: 26-36-45

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Intended useThis Calbiochem® Nitric Oxide Assay Kit, Colorimetric is intended for the spectrophotometric measurement of the nitric oxide (NO) produced in in vitro experimental systems. This method can be used to accurately measure as little as 1 µM (pmol/ml) NO in aqueous solutions and requires small quantities of samples (5 to 85 µl, depending on the concentration of nitric oxide in the sample).
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival immediately store the Nitrate Reductase at -20°C immediately and all other components at 4°C.
      Protect from Light Protect from light
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsRecombinant Zea mays Nitrate Reductase, NADH, Nitrate Standard, Color Reagents, Buffer, 96-Well Plate, and a user protocol.
      Specifications

      Documentation

      Nitric Oxide Assay Kit, Colorimetric FDS

      Titre

      Fiche de données de sécurité des matériaux (FDS) 

      Nitric Oxide Assay Kit, Colorimetric Certificats d'analyse

      TitreNuméro de lot
      482650

      Références bibliographiques

      Aperçu de la référence bibliographique
      Nims, R.W., et al. 1996. Methods Enzymol. 268, 93.

      Citations

      Titre
    • Carlos Milla, et al. (2004) Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation. American Journal of Physiology Lung Cellular and Molecular Physiology 287, L706-L714.
    • Protocole Utilisateur

      Revision28-September-2007 RFH
      Form80 Tests
      Format96-well plate
      Detection methodColorimetric
      StorageUpon arrival immediately store the Nitrate Reductase at -20°C immediately and all other components at 4°C.
      Intended useThis Calbiochem® Nitric Oxide Assay Kit, Colorimetric is intended for the spectrophotometric measurement of the nitric oxide (NO) produced in in vitro experimental systems. This method can be used to accurately measure as little as 1 µM (pmol/ml) NO in aqueous solutions and requires small quantities of samples (5 to 85 µl, depending on the concentration of nitric oxide in the sample).
      Principles of the assayIn aqueous solution, nitric oxide is rapidly converted to nitrate and nitrite (Figure 1). The ratio of the nitrate and nitrite concentrations produced from nitric oxide may vary substantially depending on the biological fluids and tissue culture media used. Hence, for accurate assay of the total nitric oxide generated, one must monitor both nitrate and nitrite levels. Spectrophotometric quantitation of nitrite using the Griess Reagent is a straightforward method; however, it does not measure nitrate. Therefore, the NADH-dependent enzyme nitrate reductase is used to convert the nitrate to nitrite prior to quantitation using the Griess Reagent --- thus providing for accurate determination of total NO produced. In addition to providing all necessary components in a microtiter format, this kit employs immunoaffinity purified Zea mays nitrate reductase and NADH, thereby circumventing some of the potential problems reported for nitric oxide measurement using NADPH dependent nitrate reductases.

      Figure 1: Assay Characteristics

      Materials provided• Nitrate Reductase: 1 vial, One unit lyophilized enzyme.
      • Nitrate Reductase Buffer: 1 vial, 1.5 ml glycerol buffer solution
      • Buffer: 1 bottle, 25 ml, 50 mM MOPS, 1 mM EDTA, pH 7.0.
      • NADH: 1 vial, 2 mg, NADH (β-Nicotinamide adenine dinucleotide, reduced form). Protect from light.
      • Color Reagent #1: 1 vial, 7 ml, Sulfanilamide (p-Aminobenzenesulfonamide) in 3 N HCl. Protect from light.
      • Color Reagent #2: 1 vial, 7 ml, N-(1-Naphthyl)ethylenediamine dihydrochloride in deionized water. Protect from light.
      • Nitrate Standard: 1 vial, 1.5 ml, 500 pmol/µl KNO₃ (equivalent to 500 µM NO).
      • 96-Well plate: One 96 well low-protein binding plate with flat bottom wells.
      • Reagent Reservoirs: Three plastic troughs for dispensing and pipetting reagents.
      Materials Required but not provided Distilled or deionized water
      Microplate reader with 540 nm filter. NOTE: The wavelength of the filter can be 530 to 560 nm, however, maximum absorbance occurs at 540 nm.
      Precision pipettes ranging from 1 µl to 100 µl, and disposable pipette tips. NOTE: If all 96 wells are to be used at one time, it is suggested that a multichannel pipettor be used.
      Clean test tubes
      Vortex mixer
      Precautions and recommendations1. Samples containing significant levels of protein (e.g., serum or tissue culture media) may produce a precipitate that may interfere with accurate measurement of NO. If a precipitate is visible after addition of Color Reagent #1, remove excess proteins (e.g. by boiling 5 min and centrifuging 10,000 x 5 min) prior to performing the assay. Alternatively, the reaction may be performed in a conical bottomed plate. Then, prior to reading absorbances, centrifuge the plate and transfer 85 µl of supernatant from each well to the corresponding well of a plate with optically clear flat-bottomed wells.
      2. For best results, complete the reading of the plate within 20 min.
      3. Color reagent #2 and the NADH solution should be kept in the dark.
      4. If the NO concentration in your sample is low, you can increase the sample volume to 85 µl by decreasing the buffer volume.
      5. 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.
      6. If additional plates are used, they should be low-protein-binding plates. If used to read absorbances, they must have optically clear, flat-bottomed wells.
      7. For a complete set of experiments, it is better to use plates obtained from the same batch to minimize errors and optical distortion.
      Reagent preparationStandards: Prepare standards by making serial dilutions as follows: A. 500 µM Nitrate standard is provided B. Take 1 ml of A, add 4 ml deionized H2O and mix = 100 µM C. Take 1 ml of B, add 1 ml deionized H2O and mix = 50 µM D. Take 1 ml of C , add 1 ml of deionized H2O and mix = 25 µM E. Take 1 ml of D, add 1.5 ml deionized H2O and mix - 10 µM F. Take 1 ml of E, add 1 ml deionized H2O and mix = 5 µM G. Take 1 ml of F, add 4 ml deionized H2O and mix = 1 µM H. Take 1 ml of G, add 1 ml deionized H2O and mix = 0.5 µM Standards can be stored at 4°C. Nitrate Reductase: Reconstitute the Nitrate Reductase in 1 ml Nitrate Reductase Buffer at room temperature for 20 min with light vortexing at 0, 10, and 20 min. Following reconstitution, aliquot and freeze (-20°C or -80°C). Reconstituted enzyme is stable for up to 6 months at -20°C or for up to 1 year at -80°C. The final concentration of enzyme is 1 U/µl. NADH: Prepare a 2 mM working solution of NADH by adding 1.28 ml deionized water to the NADH vial prior to use.
      Detailed protocol1. Determine the number of wells to be used and the organization of the samples on the microplate.

      Table 1: Sample Plate



      2. Add Standards to wells as described below:

      Table 2: Sample Standard Solutions


      3. Add 85 µl Standards and 5-85 µl samples (depending on the [NO] in the sample) to wells, in duplicate.
      4. Add sufficient Buffer to each sample to bring the volume to 85 µl (e.g., 80 µl for 5 µl sample). If the NO concentration is very low, it is possible to use 85 µl sample and no buffer.
      5. Add 10 µl of reconstituted Nitrate Reductase to each well.
      6. Add 10 µl of 2 mM NADH to each well and shake the plate for 20 min at room temperature.
      7. Add 50 µl Color Reagent #1 and shake briefly.
      8. Add 50 µl Color Reagent #2. Shake gently for 5 min at room temperature.
      9. Read absorbance at 540 nm in a plate reader. NOTE: Although the color is stable for several h, we recommend to complete reading of the plate within 20 min.
      Standard curvePlot the standard curve and calculate the concentrations of the samples from the curve. a. Subtract the average absorbance value of the blank wells (S0) from the absorbance values obtained with all the other pairs of wells. b. Average the absorbance values for each pair of duplicate wells. c. Plot a standard curve using the average absorbance value for each standard versus the concentration of standard. d. Determine the concentration of each unknown by interpolation from the standard curve.

      Figure 2: Typical Standard Curve

      Registered TrademarksCalbiochem® is a registered trademark of EMD Biosciences, Inc.
      Interactive Pathway™ is a trademark of EMD Biosciences, Inc.