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48-602MAG
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1 Kit
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Description
Overview
Assay 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 Number
482650
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
References
Nims, R.W., et al. 1996. Methods Enzymol. 268, 93.
Product Information
Detection method
Colorimetric
Form
80 Tests
Format
96-well plate
Kit contains
Recombinant Zea mays Nitrate Reductase, NADH, Nitrate Standard, Color Reagents, Buffer, 96-Well Plate, and a user protocol.
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 Phrase
S: 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 use
This 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 Conditions
Upon arrival immediately store the Nitrate Reductase at -20°C, the Microplate at room temperature and all other components at 4°C.
Protect from Light
Protect from light
Do not freeze
Ok to freeze
Supplemental Information
Kit contains
Recombinant Zea mays Nitrate Reductase, NADH, Nitrate Standard, Color Reagents, Buffer, 96-Well Plate, and a user protocol.
Carlos Milla, et al. (2004) Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation. American Journal of Physiology Lung Cellular and Molecular Physiology287, L706-L714.
User Protocol
Revision
11-December-2022 JSW
Form
80 Tests
Format
96-well plate
Detection method
Colorimetric
Storage
Upon arrival immediately store the Nitrate Reductase at -20°C, the Microplate at room temperature and all other components at 4°C.
Intended use
This 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 assay
In 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 recommendations
1. 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 preparation
Standards: 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 protocol
1. 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 curve
Plot 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 Trademarks
Calbiochem® is a registered trademark of EMD Biosciences, Inc. Interactive Pathway™ is a trademark of EMD Biosciences, Inc.
