Nitric Oxide (NO) Colorimetric Assay Kit

    • Biochemical Assay Kit-Elabscience
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    • Biochemical Assay Kit-Elabscience
    • Biochemical Assay Kit-Elabscience

      Catalog number:E-BC-K036

      Size:
      • 48T
      • 96T
      Qty:
      - +
      Price: $120

      Detection method: Colorimetric method

      Detection instrument: Microplate reader

      Valid period: 6 months

      Lead Time: 7~10 daysWelcome to order from local distributors.

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      Application

      The kit applies to detect the concentration of NO in serum, plasma, tissue, cells, cell culture supernatant and other samples. 


      Detection significance

      The half-life of NO is extremely short, it exists in form of nitrate or nitrite produced by vascular endothelial cell, vascular smooth muscle cell, platelet, and macrophage and so on. The concentration of NO can be indirectly measured by detecting that of nitrate or nitrite.

      NO react with oxygen and water to generate nitrate or nitrite which can form a kind of pale red azo compound when meet with nitrate chromogenic reagent, the absorbance of the compound can be measured to calculate the concentration of NO indirectly.


      Detection principle

      NO is easily oxidized to form NO2- in vivo or in aqueous solution, and a reddish azo compound is formed with the color developing agent, and the concentration of the azo compound is linearly related to the concentration of NO. The concentration of NO can be calculated indirectly by measuring the OD value at 550 nm. 

                                             


      Experiment instruments

      Tubes, micropipette, vortex mixer, microplate reader (550 nm)


      Sample preparation

      1. Tissue homogenates: Mince the tissues to small pieces, then be weighed and homogenized in homogenization medium (0.01M PBS, pH 7.4) on ice, the volume of PBS (mL): the weight of the tissue (g) =9:1. The tissue homogenate is centrifuged for 10 min at 3100 g and collect the supernatant for detect. Meanwhile, determine the concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

      2. Serum (plasma): detect directly.

      3. Cultured cells:

            Collect the cells and wash the cells with homogenization medium (0.01M PBS, pH 7.4) for 1~2 times. Centrifuge at 1000 g for 10 min and then discard the supernatant and keep the cell sediment. Add PBS at a ratio of cell number (106): PBS (μL) =1: 300-500. Sonicate or grind with hand-operated in ice water bath. Centrifuge at 3100 g for 10 min, then take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

      4. Cell culture supernatant:

      Detect directly. Centrifuge at 3100 g for 10 min if there is turbidity, then take the supernatant and preserve it on ice for detection.


      Operation procedure

      1. Dilution of standard

      Dilute 2 mmol/L standard sodium nitrite solution with deionized water to a serial concentration. The recommended dilution gradient is as follows: 300, 200, 150, 100, 40, 20, 10, 0 μmol/L.

      2. Pre-treatment

       

      Standard tube

      Sample tube

      Different concentrations of sodium nitrite (μL)

      A*

       

      Sample (μL)

       

      A*

      Reagent 1 (μL)

      200

      200

      Reagent 2 (μL)

      100

      100

      Mix fully and stand for 15 min at room temperature, centrifuge it at 3100 g for 10 min, take 160 μL of the supernatant for the following procedure.

      Note: a: For serum or plasma samples, A* is 200-300 μL.

                 b: For tissue or cell homogenates, A* is 100-300 μL.

      3. Reaction

       

      Standard tube

      Sample tube

      Supernatant (μL)

      160

      160

      Chromogenic reagent (μL)

      80

      80

      Mix thoroughly for 2 min, stand for 15 min at room temperature, measure the OD of each well with micro-plate reader immediately at 550 nm wavelength.



      Technical parameter

      1. The sensitivity of the kit is 0.16 μmol/L.

      2. The intra CV is 2.4% and the inter CV is 3.7%.

      3. The recovery of the kit is 102%.

      4. The detection range of the kit is 0.16~300 μmol/L.


      Notes

      1. The kit is for scientific research only.

      2. Instructions should be followed strictly, changes of operation may result in unreliable results.

      3. The validity of kit is 6 months.

      4. Do not use components from different batches of kit.

      5. Use disposable EP tubes or clean glass tubes with stopper for centrifugation.

      6. The supernatant for assay should not contain sediment, otherwise it will affect the results.

      7. Samples can be stored at -20 for 1-2 months, the lower the temperature, the longer the storage time.

      8. All reagents should be prepared the day before the assay, let it fully dissolved. Please add reagents to the bottom of well vertically and slowly, avoid to add on the wall of well and generate bubble. 





      Citations

      1. Publication: SONG Quan Quan, NIU Jing Ping, ZHANG Shu Yu, LIANG Ting Ting, ZHOU Ji, FENG Shan Shan. Effects of Simulated Heat Wave and Ozone on High Fat Diet ApoE Deficient Mice. Biomedical and Environmental Sciences, 2018, 31(10): 757-768.
        Sample Type: Mouse
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