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Nitric Oxide Synthase Assay Kit

AcA
产品编号 ABIN1000324
发货至: 中国
  • 抗原 See all Nitric Oxide Synthase (NOS) 试剂盒
    Nitric Oxide Synthase (NOS)
    应用范围
    Activity Assay (AcA)
    特异性
    0.25 U/L
    产品特性
    Sensitive and accurate. Detection range 0.25 - 25 U/L in 96-well plate.
    Rapid and reliable. Can be completed in 40 min if reduction of NO3 - to NO2 - is performed at 60°C.
    组件
    Assay Buffer: 10 mL. Substrate: 600 µL. GDH: 120 µL. Reagent A: 12 mL. Reagent B: 500 µL. Reagent C: 12 mL. Reagent D: 600 µL. Reagent E: 1.5 mL. ZnSO4: 1 mL. Standard: 1 mL. NaOH: 1 mL.
    试剂未包括
    Pipetting devices, eppendorf tubes, eppendorf centrifuge, clear, flat bottomed 96 well plates or cuvettes, plate reader or spectrophotometer and heat block or hot water bath (optional).
    Top Product
    Discover our top product NOS ELISA Kit
  • 应用备注
    Direct Assays: NOS activity in biological samples.
    Drug Discovery/Pharmacology: effects of drugs on NOS activity.
    说明

    Antioxidants and nucleophiles (e.g. beta-mercaptoethanol, glutathione, dithiothreitol and cysteine) may interfere with this assay. Avoid using these compounds during sample preparation. However, if beta-mercaptoethanol or dithiothreitol must be used, an equal concentration needs to be added to the standards.

    实验流程
    Prior to assay, equilibrate all components to room temperature. Prewarm Assay Buffer to 37°C. Keep GDH on ice. Sample treatment: tissue or cell samples are homogenized in 1 x PBS (pH 7.4). Centrifuge at 10,000g or higher at 4°C. Use supernatant for NOS assay. Standard preparation: Prepare 200 µL 500 µMPremix by mixing 100 µL 1.0 mM Standard and 100 µL distilled water. Dilute standards in1.5- mL centrifuge tubes.
    NOS Reaction: If samples will not require deproteination (i.e. purified NOS), add 20 µL of each sample and standard to separate labeled eppendorf tubes. Each sample requires at least two tubes: one reaction tube and one sample blank tube. Immediately prior to starting the reaction, prepare enough NOS Working Reagent (NOS WR) for all sample reaction tubes and standards by mixing per reaction tube: 65 µL Assay Buffer, 4 µL Substrate, 4 µL Reagent D, 10 µL Reagent E and 1 µL GDH. For the sample blanks, use 8 µL dH2O instead of the Substrate and Reagent D. Add 80 µL of the appropriate NOS WR to each tube and incubate at 37°C for 20 min. After 20 min immediately add 200 µL of the NO Detection Reagent (NO DR) (see next section: NO Measurement) to each tube to kill the NOS reaction. For samples requiring deproteination which include serum, plasma, whole blood, cell culture media containing FBS, tissue or cell lysates, add 25 µL of each sample and standard to separate labeled eppendorf tubes. Each sample requires at least two tubes: one reaction tube and one sample blank tube. Immediately prior to starting the reaction, prepare enough NOS WR for all sample reaction tubes and standards by mixing per reaction tube: 80 µL Assay Buffer, 5 µL Substrate, 5 µL Reagent D, 13 µL Reagent E and 1 µL GDH. For the sample blanks, use 10 µL dH2O instead of the Substrate and Reagent D. Add 100 µL of the appropriate NOS WR to each tube and incubate at 37°C for 20 min. After 20 min immediately proceed to the deproteination step. Deproteination. Add 7 µL ZnSO4 to each sample and standard tube. Vortex and then add 7 µL NaOH. Vortex again and centrifuge 10 min at 14,000 rpm. Transfer 100 µL of the clear supernatant to a clean tube and proceed to the NO Measurement step.
    NO Measurement: Immediately prior to starting the reaction, prepare enough NO Detection Reagent (NO DR) for all samples and standards by mixing per reaction tube: 100 µL Reagent A, 4 µL Reagent B and 100 µL Reagent C. Add 200 µL of the WR to each sample and standard tube and incubate for 5 min at 60°C. (Alternatively, the reaction can be run at 37°C for 60 min or RT for 150 min.) Briefly centrifuge the reaction tubes to pellet any condensation and transfer 250 µL of each reaction to separate wells in a 96 well plate. Read OD at 500-570nm (peak 540 nm).
    结果分析

    Subtract blank OD (Std 4) from the standard OD values and plot the OD against standard concentrations.

    限制
    仅限研究用
  • 储存条件
    4 °C
  • Meng, Ganesan Adaikan, Srilatha: "Hydrogen sulfide promotes nitric oxide production in corpus cavernosum by enhancing expression of endothelial nitric oxide synthase." in: International journal of impotence research, Vol. 25, Issue 3, pp. 86-90, (2013) (PubMed).

  • 抗原
    Nitric Oxide Synthase (NOS)
    别名
    Nitric Oxide Synthase (NOS 产品)
    别名
    2310005C01Rik Kit, NO Kit, NOS-I Kit, Nos-1 Kit, bNOS Kit, nNOS Kit, CG6713 Kit, DNOS Kit, DNOS1 Kit, Dmel\\CG6713 Kit, NOS Kit, dNOS Kit, dNos Kit, drNOSoxy Kit, GB18010 Kit, Nos Kit, BmNOS Kit, iNOS-LP Kit, NOS1 Kit, nitric oxide synthase 1, neuronal Kit, Nitric oxide synthase Kit, nitric oxide synthase Kit, nitric oxide synthase 1 Kit, neuropeptide S Kit, Nos1 Kit, Nos Kit, NOS Kit, NOS1 Kit, NPS Kit
    背景
    Quantitative determination of nitric oxide synthase activity by colorimetric (540nm) method.
    Procedure: 40 min.

    Nitric oxide (NO) is a reactive radical that plays an important role in many key physiological functions. NO, an oxidation product of arginine by nitric oxide synthase (NOS), is involved in host defense and development, activation of regulatory proteins and direct covalent interaction with functional biomolecules. Simple, direct and non-radioactive procedures for measuring NOS are becoming popular in Research and Drug Discovery. This Nitric Oxide Synthase Assay Kit involves two steps: a NOS reaction step during which NO is produced followed by an NO detection step. Since the NO generated by NOS is rapidly oxidized to nitrite and nitrate, the NO production is measured following reduction of nitrate to nitrite using an improved Griess method. The procedure is simple and the time required for sample pretreatment and assay is reduced to as short as 40 min.
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