Nitric Oxide Synthase Assay Kit, BioAssay™
Référence N2577-40-96T
Conditionnement : 96Tests
Marque : US Biological
Contactez votre distributeur local :
Téléphone : +1 850 650 7790
Shipping Temp
Dry IceStorage Temp
4°C/-20°CNitric 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. 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.
Key Features:
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.
Rapid and reliable. Can be completed in 40 min if reduction of NO3- to NO2- is performed at 60°C.
Applications:
Direct Assays: NOS activity in biological samples.
Drug Discovery/Pharmacology: effects of drugs on NOS activity.
Drug Discovery/Pharmacology: effects of drugs on NOS activity.
Kit Contents (100 tests in 96-well plates):
Assay Buffer: 10ml Substrate: 600ul GDH: 120ul
Reagent A: 12ml Reagent B: 500ul Reagent C: 12ml
Reagent D: 600ul Reagent E: 1.5ml ZnSO4: 1ml
Standard: 1ml N aOH: 1ml
Storage conditions. The kit is shipped on ice. Store Assay Buffer, Substrate, Reagent D, Reagent E and GDH at -20°C. Store all other reagents at 4°C. Shelf life of three months after receipt. Precautions: reagents are for research use only. Please refer to Material Safety Data Sheet for detailed information.
Reagent A: 12ml Reagent B: 500ul Reagent C: 12ml
Reagent D: 600ul Reagent E: 1.5ml ZnSO4: 1ml
Standard: 1ml N aOH: 1ml
Storage conditions. The kit is shipped on ice. Store Assay Buffer, Substrate, Reagent D, Reagent E and GDH at -20°C. Store all other reagents at 4°C. Shelf life of three months after receipt. Precautions: reagents are for research use only. Please refer to Material Safety Data Sheet for detailed information.
Procedures:
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 200ul 500uM Premix by mixing 100ul 1.0mM Standard and 100ul distilled water. Dilute standards in 1.5-mL centrifuge tubes as described in the Table.
No Premix+H2O Nitrite (uM)
1 50ul+0ul 500
2 30ul+20ul 300
3 15ul+35ul 150
4 0ul+50ul 0
NOS Reaction: If samples will not require deproteination (i.e. purified NOS), add 20ul 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: 65ul Assay Buffer, 4ul Substrate, 4ul Reagent D, 10ul Reagent E and 1ul GDH. For the sample blanks, use 8ul dH2O instead of the Substrate and Reagent D. Add 80ul of the appropriate NOS WR to each tube and incubate at 37°C for 20 min. After 20 min immediately add 200ul 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 25ul 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: 80ul Assay Buffer, 5ul Substrate, 5ul Reagent D, 13ul Reagent E and 1ul GDH. For the sample blanks, use 10ul dH2O instead of the Substrate and Reagent D. Add 100ul 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 7ul ZnSO4 to each sample and standard tube. Vortex and then add 7ul NaOH. Vortex again and centrifuge 10 min at 14,000 rpm. Transfer 100ul 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: 100ul Reagent A, 4ul Reagent B and 100ul Reagent C. Add 200ul 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 250ul of each reaction to separate wells in a 96 well plate. Read OD at 500-570nm (peak 540 nm).
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 200ul 500uM Premix by mixing 100ul 1.0mM Standard and 100ul distilled water. Dilute standards in 1.5-mL centrifuge tubes as described in the Table.
No Premix+H2O Nitrite (uM)
1 50ul+0ul 500
2 30ul+20ul 300
3 15ul+35ul 150
4 0ul+50ul 0
NOS Reaction: If samples will not require deproteination (i.e. purified NOS), add 20ul 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: 65ul Assay Buffer, 4ul Substrate, 4ul Reagent D, 10ul Reagent E and 1ul GDH. For the sample blanks, use 8ul dH2O instead of the Substrate and Reagent D. Add 80ul of the appropriate NOS WR to each tube and incubate at 37°C for 20 min. After 20 min immediately add 200ul 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 25ul 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: 80ul Assay Buffer, 5ul Substrate, 5ul Reagent D, 13ul Reagent E and 1ul GDH. For the sample blanks, use 10ul dH2O instead of the Substrate and Reagent D. Add 100ul 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 7ul ZnSO4 to each sample and standard tube. Vortex and then add 7ul NaOH. Vortex again and centrifuge 10 min at 14,000 rpm. Transfer 100ul 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: 100ul Reagent A, 4ul Reagent B and 100ul Reagent C. Add 200ul 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 250ul of each reaction to separate wells in a 96 well plate. Read OD at 500-570nm (peak 540 nm).
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 NOS activity of the Sample is then calculated as
ODSAMPLE-ODBLANK
Slope
NOS Activity=X (U/L)
1
t
ODSAMPLE and ODBLANK are optical density values of the sample and sample blank, respectively. t is the reaction time (20 min).
Unit definition: one unit of NOS catalyzes the production of 1uMole of nitric oxide per minute under the assay conditions (pH 7.5 and 37°C).
ODSAMPLE-ODBLANK
Slope
NOS Activity=X (U/L)
1
t
ODSAMPLE and ODBLANK are optical density values of the sample and sample blank, respectively. t is the reaction time (20 min).
Unit definition: one unit of NOS catalyzes the production of 1uMole of nitric oxide per minute under the assay conditions (pH 7.5 and 37°C).
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. However, if b-mercaptoethanol or dithiothreitol must be used, an equal concentration needs to be added to the standards.
Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.