482655 Nitric Oxide Assay Kit, Fluorometric

482655
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Overview

Replacement Information

Key Specifications Table

Detection Methods
Fluorometric

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      Description
      OverviewAssay 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.
      Catalogue Number482655
      Brand Family Calbiochem®
      Materials Required but Not Delivered A fluorometric plate reader capable of measuring fluorescence (excitation = 365 nm; emission = 450 nm)
      An adjustable pipette
      Glass distilled or HPLC-Grade water
      References
      ReferencesMiles, 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.
      Product Information
      Detection methodFluorometric
      Form192-288 Tests
      Format96-well plate
      Kit containsAssay Buffer, Nitrate Reductase, Enzyme Cofactors, Nitrate Standard, Nitrite Standard, DAN Reagent, NaOH, Microtiter Plates, Plate Covers, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time3-5 h
      Sample TypeAqueous solutions
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 34-36/37/38-45

      Causes burns.
      Irritating to eyes, respiratory system and skin.
      May cause cancer.
      S PhraseS: 26-36/37/39-45-53

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Avoid exposure - obtain special instructions before use.
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: 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.
      Storage -20°C
      Storage ConditionsUpon arrival, store the entire contents of the kit at -20°C until use. For storage information on individual components following initial thawing and reconstitution, please consult the section on Pre-Assay Preparation.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAssay Buffer, Nitrate Reductase, Enzyme Cofactors, Nitrate Standard, Nitrite Standard, DAN Reagent, NaOH, Microtiter Plates, Plate Covers, and a user protocol.
      Specifications

      Documentation

      Nitric Oxide Assay Kit, Fluorometric SDS

      Title

      Safety Data Sheet (SDS) 

      Nitric Oxide Assay Kit, Fluorometric Certificates of Analysis

      TitleLot Number
      482655

      References

      Reference overview
      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.
      User Protocol

      Revision09-March-2010 JSW
      Form192-288 Tests
      Format96-well plate
      Detection methodFluorometric
      StorageUpon arrival, store the entire contents of the kit at -20°C until use. For storage information on individual components following initial thawing and reconstitution, please consult the section on Pre-Assay Preparation.
      BackgroundNitric oxide (NO), produced in trace quantities by neurons, endothelial cells, platelets, neutrophils and other cells, acts as a unique second messenger molecule. It readily diffuses through cell membranes to exert a variety of biological actions in mammalian cells. Excess generation of NO lead to the formation of peroxynitrite, destruction of iron-sulfur clusters, thiol nitrosylation, and nitration of protein tyrosine residues. The final products of NO in vivo are nitrite (NO2-) and nitrate (NO3-). The relative proportions of these two products are variable. Hence, the best index of total NO produced is the sum of both [NO2-] and [NO3-].
      Principles of the assayThe Calbiochem° Nitric Oxide Assay Kit, Fluorometric provides an accurate and convenient method for the measurement of total [NO₂⁻] and [NO₃⁻] in a simple two-step process. The first step involves the conversion of nitrate to nitrite by the enzymatic action of nitrate reductase. The second step involves the addition of 2,3-diaminonaphthalene (DAN) followed by NaOH to convert nitrite to a fluorescent compound 1(H)-naphthotriazole. Measurement of fluorescence of 1(H)-naphthotriazole provides an accurate assay of [NO₂⁻]

      Figure 1: Principle of the Assay

      Materials provided• Assay buffer (Kit Component No. KP0201): 1 vial
      • Nitrate reductase (Kit Component No. KP0202): 2 vials
      • Enzyme co-factors (Kit Component No. KP0203): 2 vials
      • Nitrate standard (Kit Component No. KP0204): 1 vial
      • Nitrite Standard (Kit Component No. KP0205): 1 vial
      • DAN reagent (Kit Component No. KP0206): 1 vial
      • NaOH (2.8 M (Kit Component No. KP0207): 1 vial
      • 96-well plate and Covers(Kit Component No. KP0208): 3 plates, 3 plate covers
      Materials Required but not provided A fluorometric plate reader capable of measuring fluorescence (excitation = 365 nm; emission = 450 nm)
      An adjustable pipette
      Glass distilled or HPLC-Grade water
      Precautions and recommendations Useful Pipetting Tips
      a) To maintain precise times of incubation and for saving time, use of a repipettor is recommended.
      b) Always use different tips for pipetting assay buffer, standards, samples and color development reagents.
      c) Before pipetting, equilibrate the pipette tip in the reagent to be used (i.e., fill the tip and expel the solution, repeat a couple of times).
      d) Do not touch the pipette tip to the reagents already in the well.
      Preparation1. 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: Homogenize the sample in phosphate-buffered saline (PBS, pH 7.4) and centrifuge at 10,000 x g for 20 min. Centrifuge at 100,000 x g for 30 min (this second centrifugation is optional, but will increase filtration rates). Ultrafilter the supernatant through a 10 or 30 kDa cut-off filter. Use 10 µl of the filtrate for nitrate and/or nitrite assay. The conversion of nitrate to nitrite requires about 2 h for ≥95% conversion.
      Reagent preparation1. Assay Buffer: Dilute the contents of the vial to 100 ml with HPLC-Grade water. Use this buffer for diluting samples, as needed, prior to assay. Store at 4°C. This buffer will be stable for ~2 months at 4°C. 2. Nitrate Reductase: Reconstitute the contents of vial with 1.2 ml of assay buffer. Keep on ice during use. Aliquot and store at -20°C. Allow only one time freezing and thawing of this solution. 3. Enzyme Co-factors: Reconstitute the contents of this vial with 1.2 ml of assay buffer. Keep on ice during use. Aliquot and store at -20°C. Allow only one time freezing and thawing of this solution. 4. Nitrate Standard: Remove the vial stopper slowly to minimize disturbance of the lyophilized powder. Reconstitute the lyophilized nitrate standard using 1.0 ml of Assay Buffer. The concentration of this solution is 2 mM. Swirl to ensure that powder clinging to the sides of the vial is dissolved. Vortex gently. Store all stock solutions at 4°C; do not freeze after reconstitution. When stored under these conditions, the nitrate standard is stable for at least three months. 5. Nitrite Standard: Remove the vial stopper slowly to minimize disturbance of the lyophilized powder. Reconstitute the lyophilized nitrite standard using 1 ml of Assay Buffer. The concentration of this solution is 2 mM. Swirl to ensure that powder clinging to the sides of the vial is dissolved. Vortex gently. Store all stock solutions at 4°C; do not freeze after reconstitution. When stored under these conditions, the nitrite standard is stable for at least three months. 6. Fluorometric reagent (DAN): Ready to use. Store at 4°C. Do not add water or assay buffer to this vial. 7. NaOH : Ready to use. Store at 4°C. Do not add water or assay buffer to this vial.
      Detailed protocolA. Measurement of Nitrate + Nitrite

      1. Preparation of Nitrate Standard Curve: A nitrate standard curve must be performed in order to quantitate sample nitrate + nitrite concentrations. In a clean test tube, place 900 µl of Assay Buffer. To this, add 100 µl of reconstituted nitrate standard and vortex. Use this diluted standard (200 µM) for the preparation of the nitrate standard curve as described below.

      Obtain 8 clean test tubes and label them as 1 through 8. Aliquot 950 µl of assay buffer to tube 1 and 500 µl of assay buffer to tubes 2 through 8. Transfer 50 µl of nitrate standard into test tube 1 and mix well. The concentration of standard in tube 1 is 10 µM. Serially dilute the nitrate standard by removing 500 µl volume from tube 1 and placing in tube 2. Mix well. Then remove 500 µl from tube 2 and place it in tube 3. Repeat the procedure with tubes 4 to 7. Mix well after each addition. DO NOT store the diluted standards for more than 1-2 h.

      Table 1: Measurement of Nitrate + Nitrite


      2. Aliquoting the Standards for the Standard Curve: Reserve nine wells on the plate for each standard curve (for better data we recommend running standards in duplicate) [Note: if you use a single cell spectrofluorometer, perform all reactions in small test tubes.] Add 80 µl of the assay buffer to the first standard well and 30 µl to each of the remaining eight. Add 50 µl of nitrate standard from test tube #8 to the second standard well on the plate. Then add 50 µl from test tube #7 to the third standard well. Continue this process for test tube #6, 5,4,3,2, and 1.
      3. Aliquoting the Samples: Add 10-20 µl of sample to the wells and adjust the volume to 80 µl with Assay Buffer. [Note: For plasma samples and tissue homogenates, use no more than 10 µl of undiluted plasma or filtrate per well.] Avoid any bubbles from entering the wells.
      4. Aliquoting the Enzyme Co-Factors: Add 10 µl of the enzyme co-factors to each well.
      5. Aliquoting the Nitrate Reductase: Add 10 µl of the nitrate reductase to each well.
      6. Incubation: Cover the plate with the plate cover and incubate at room temperature for 30 min. This incubation period should be increased to 1 h when assaying tissue culture medium or 2 h when assaying plasma and tissue samples.
      7. Aliquoting DAN: After the required incubation period add 10 µl of DAN reagent to each well and incubate for 10 min.
      8. Aliquoting NaOH: Add 20 µl of NaOH to each well.
      9. Reading the Plate: Read the plate in a fluorometer using the excitation wavelength of 375 nm and an emission wavelength of 415 nm. Alternatively, excitation and emission wavelengths of 360-365 and 430-450 nm, respectively, can be used. (Any emission wavelength above 450 nm cannot be used.) When using a single cell spectrofluorometer, dilute the sample to 2 to 3 ml to allow for the measurement of all the samples. Higher concentrations of nitrate and nitrite may require lower gain setting, whereas the gain may need to be increased for low concentrations of nitrate and nitrite.

      B. Measurement of Nitrite

      1. Preparation of Nitrite Standard Curve and Samples: Follow the nitrate standard curve preparation instructions (see above) using the nitrite standard vial. If using a single cell fluorometer, perform all reactions in small test tubes.
      2. Aliquoting the Standards for the Standard Curve: Reserve nine wells on the plate for each standard curve (for better data we recommend running standards in duplicate) [Note: if you use a single cell spectrofluorometer, perform all reactions in small test tubes.] Add 100 µl of the assay buffer to the first standard well and 50 µl to each of the remaining eight. Add 50 µl of nitrate standard from test tube #8 to the second standard well on the plate. Then add 50 µl from test tube #7 to the third standard well. Continue this process for test tube #6, 5,4,3,2, and 1.
      3. Aliquoting the Samples: Add 10-20 µl of sample to the wells and adjust the volume to 100 µl with Assay Buffer. [Note: For plasma samples and tissue homogenates, use no more than 10 µl of undiluted plasma or filtrate per well.] Avoid any bubbles from entering the wells.
      4. Aliquoting DAN: After the required incubation period add 10 µl of DAN reagent to each well and incubate for 10 min.
      5. Aliquoting NaOH: Add 20 µl of NaOH to each well.
      6. Reading the Plate: Read the plate in a fluorometer using the excitation wavelength of 365 nm and an emission wavelength of 450 nm. Alternatively, excitation and emission wavelengths of 375 and 415 nm, respectively, can be used. Do not use emission wavelengths above 450 nm. When using a single cell spectrofluorometer, dilute the sample to 2 to 3 ml to allow for the measurement of all the samples. Higher concentrations of nitrate and nitrite may require lower gain setting, whereas the gain may need to be increased for low concentrations of nitrate and nitrite.
      Standard curvePlotting the Standard Curve: Make a plot of fluorescence vs. picomoles of nitrate or nitrite. The nitrate standard curve is used for determination of total nitrate plus nitrite concentration, whereas the nitrite standard curve is used for the determination of nitrite alone. In theory, these two standard curves should be identical, however, in practice a small discrepancy is often observed. Fluorescence measurements have the advantage of measuring a broad linear range. Hence, the standard curve is prepared using serial dilutions of a stock standard. It may be necessary to expand or reduce the scale in instances where extremely low or high levels or nitrate and/or nitrite are measured. A typical standard curve is shown below:

      Figure 2: Determination of Sample Nitrate or Nitrite Concentration

      Sensitivity NotesThis fluorometric assay will detect as little as 30 nM nitrite in the final reaction mixture (<4 pmol in 120 µl). When using 20 µl sample, the detection limit for nitrite in the original sample is 0.2 µM.
      Plate configurationThere is no specific recommended pattern for using the wells on the plate. However, nine wells will be required for the standard curve. For NO assay, when using tissue culture medium, the standard curve should also be prepared in the presence of the medium. If you plan to measure the total NO products (nitrate + nitrite), only the nitrate standard curve is required. If you wish to measure nitrite, then only the nitrite standard curve is needed. The remaining wells can be used for assay of samples.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.