Amplite® Fluorimetric Extracellular Nitric Oxide (NO) Activity Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Platform
Fluorescence microplate reader
Excitation | 570 nm |
Emission | 610 nm |
Cutoff | 590 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare nitric oxide working solution (50 µL)
- Add nitric oxide standard or test samples (50 µL)
- Incubate at room temperature for 30 - 60 minutes
- Monitor the fluorescence intensity
Important notes
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
- DAW-J2 stock solution (200X):
Add 25 mL of DMSO into the vial of DAW-J2 (Component A) to make 200X stock solution. - Nitric oxide standard solution (100 mM, not provided):
We used DEA NONOate (Cayman Chemical, Item No. 82100, CAS#372965-00-9) as the nitric oxide standard. The standard solution of nitric oxide was prepared at the concentration of 100 mM in 0.01N NaOH.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/16365
Add 10 µL of 100 mM NONOate standard solution into 990 uL Assay Buffer (Component B) to generate 1 mM Nitric Oxide standard solution (SD7). Then perform 1:2 serial dilutions to get serially diluted nitric oxide standards (SD6-SD1). Note: Diluted NONOate standard solution is unstable, and should be used promptly.
PREPARATION OF WORKING SOLUTION
Add 25 µL of DAW-J2 200X stock solution into 5 mL of Assay Buffer (Component B) and mix well. Note: This nitric oxide working solution is enough for 100 assays. The working solution is not stable, use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table1: Layout of nitric oxide standards and test samples in a solid black 96-well microplate. SD=Standard, BL=Blank Control, TS=Test Sample.
BL | BL | TS | TS |
SD1 | SD1 | …. | …. |
SD2 | SD2 | ||
SD3 | SD3 | ||
SD4 | SD4 | ||
SD5 | SD5 | ||
SD6 | SD6 | ||
SD7 | SD7 |
Table2: Reagent composition for each well.
Well | Volume | Reagent |
SD1-SD7 | 50 µL | Serial Dilution (15.6 to 1000 µM) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | Test Sample |
- Prepare nitric oxide standards (SD), blank controls (BL) and test samples (TS) into a solid black 96-well microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL per well.
- Add 50 µL of nitric oxide working solution to each well of standards, blank controls and test samples to make the total nitric oxide assay volume of 100 µL/well. For a 384-well plate, use 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 30 minutes to 1 hour, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 570 nm, Emission = 610 nm (cutoff = 590 nm).
Images
Citations
Authors: Luo, Zhen and Zhao, Qin and Liu, Jixiang and Liao, Jinfang and Peng, Ruogu and Xi, Yunting and Diwu, Zhenjun
Journal: Analytical biochemistry (2017): 44--48
Authors: Adamiak, Mateusz and Abdelbaset-Ismail, Ahmed and Moore, Joseph B and Zhao, J and Abdel-Latif, Ahmed and Wysoczynski, Marcin and Ratajczak, Mariusz Z
Journal: Stem Cell Reviews and Reports (2016): 1--12
References
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Journal: PLoS One (2011): e25335
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Journal: Am J Physiol Heart Circ Physiol (2011): H1788
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Journal: J Neurosci (2010): 13254
Authors: Gupta KJ, Kaiser WM.
Journal: Plant Cell Physiol (2010): 576
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Journal: ACS Chem Neurosci (2010): 182
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