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#D2NO-100 QuantiChrom™ Nitric Oxide Assay Kit
Product name : QuantiChrom™ Nitric Oxide Assay Kit
Catalog number : D2NO-100
Supplier name : Bioassays
Data sheet: Ask more or other datasheet now !
About this Product :QuantiChrom™ Nitric Oxide Assay Kit antibody storage GENTAUR recommends for long therm storage to freeze at -24 C. For short time storage up to 30 days we suggest fridge storage at 1 to 10 C. Prevent multiple freeze taw cycles of QuantiChrom™ Nitric Oxide Assay Kit.
More Details about
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, is involved in host defense and development, activation of regulatory proteins and direct covalent interaction with functional biomolecules. Simple, direct and automation-ready procedures for measuring NO are becoming popular in Research and Drug Discovery. Since NO is oxidized to nitrite and nitrate, it is common practice to quantitate total
- as a measure for NO level. BioAssay Systems'
QuantiChromTM Nitric Oxide Assay Kit is designed to accurately measure NO production following reduction of nitrate to nitrite using improved Griess method. The procedure is simple and the time required for sample pretreatment and assay is reduced to as short as 30 min.
Sensitive and accurate. Detection range 0.6 - 200 μM in 96-well plate.
Rapid and reliable. Using an optimized VCl3 reagent, the time required for reduction of
- to NO2
is 10 min at 60°C.
Simple and high-throughput. The procedure involves mixing sample with three reagents, incubation for 10 min at 60°C and reading the optical density. Can be readily automated to measure thousands of
samples per day.
Direct Assays: NO in plasma, serum, urine, tissue/cells and foods.
Drug Discovery/Pharmacology: effects of drugs on NO metabolism.
KIT CONTENTS (100 tests in 96-well plates)
Reagent A: 12 mL R eagent B: 500 μL R e a g ent C: 12 mL
NaOH: 1 mL Z n S O4: 1 mL S tandard: 1 mL
Storage conditions. The kit is shipped at room temperature. Store Reagents A, B, C and Nitrite standard at 4°C. All other components can be stored at room temp. Shelf life of six months after receipt.
Precautions: reagents are for research use only. Please refer to Material Safety Data Sheet for detailed information.
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 NO assay.
Samples that need deproteination include serum, plasma, whole blood, cell culture media containing FBS, tissue or cell lysates. Urine and saliva do not need deproteination.
Deproteination. Mix 150 μL sample with 8 μL ZnSO4 in 1.5-mL tubes.
Vortex and then add 8 μL NaOH, votex again and centrifuge 10 min at 14,000 rpm. Transfer 100 μL of the clear supernatant to a clean tube.
Note: If samples need to be deproteinated, 150 μL of each standard should be prepared and also treated with ZnSO4 and NaOH to eliminate the need for a dilution factor.
Procedure using 96-well plate:
1. Standards. Prepare 500 μL 100 μM Premix by mixing 50 μL 1.0 mM
Standard and 450 μL distilled water. Dilute standards in 1.5-mL
centrifuge tubes as described in the Table.
2. Reaction. Add 100 μL of each sample to separate, labeled eppendorf tubes. (We recommend that samples be measured in at least duplicate). Immediately prior to starting the reaction, prepare enough
Working Reagent (WR) 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 10 min at 60°C. (Alternatively, the reaction can be run at 37°C for 60 min or RT for 150 min.)
3. Measurement. 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).
Procedure using Cuvette:
Prepare standards and samples as described for the 96-well procedure except quadruple (4×) the volumes. After the reaction, transfer 1 mL to a cuvette. Measure OD540nm in the cuvette.
Subtract blank OD (Std 4) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The NO concentration of Sample is calculated as
ODSAMPLE and ODBLANK are optical density values of the sample and water, respectively.
Conversions: 1 mg/dL NO equals 333 μM, 0.001% or 10 ppm.
MATERIALS REQUIRED, BUT NOT PROVIDED
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).
Antioxidants and nucleophiles (e.g. b-mercaptoethanol, glutathione, dithiothreitol and cysteine) may interfere with this assay. Avoid using these compounds during sample preparation.
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