Nitric Oxide Assay Kit, BioAssay™

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 NO2-/NO3- as a measure for NO level. 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.||Key Features:|Sensitive and accurate. Detection range 0.6-200uM in 96-well plate.|Rapid and reliable. Using an optimized VCl3 reagent, the time required for reduction of NO3- 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.||Applications:|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: 12ml Reagent B: 500ul Reagent C: 12ml|NaOH: 1ml ZnSO4: 1ml Standard: 1ml|Storage conditions. The kit is shipped at room temperature. Store all reagents at 2-8°C. Shelf life of six months after receipt.|Precautions: reagents are for research use only. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|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 150ul sample with 8ul ZnSO4 in 1.5-mL tubes. Vortex and then add 8ul NaOH, votex again and centrifuge 10 min at 14,000 rpm. Transfer 100ul of the clear supernatant to a clean tube.|Note: If samples need to be deproteinated, 150ul 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 500ul 100uM Premix by mixing 50ul 1.0mM Standard and 450ul distilled water. Dilute standards in 1.5-mL centrifuge tubes as described in the Table.|No Premix+H2O Nitrite (uM)|1 250ul+0ul 100|2 150ul+100ul 60|3 75ul+175ul 30|4 0ul+250ul 0|2. Reaction. Add 100ul 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: 100ul Reagent A, 4ul Reagent B and 100ul Reagent C. Add 200ul 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 250ul 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 1ml to a cuvette. Measure OD540nm in the cuvette.||Calculation:|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|[Nitric Oxide]=ODSAMPLE-ODBLANK|Slope (uM)|ODSAMPLE and ODBLANK are optical density values of the sample and water, respectively.|Conversions: 1 mg/dL NO equals 333uM, 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).||General Considerations:|Antioxidants and nucleophiles (e.g. b-mercaptoethanol, glutathione, dithiothreitol and cysteine) may interfere with this assay. Avoid using these compounds during sample preparation.