Amplite® Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*
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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 |
Excitation (nm) | 571 |
Emission (nm) | 584 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | SDSProtocol |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540nm |
Emission | 590nm |
Cutoff | 570nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- XO standards or test samples (50 µL)
- Add XO working solution (50 µL)
- Incubate at room temperature for 15 - 30 minutes
- Read fluorescence intensity at Ex/Em = 540/590 nm (cut off 570 nm)
Important notes
Thaw all the kit components to room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Amplite™ Red stock solution (250X):
Add 40 µL of DMSO (Component F) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Note: The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH = 7 – 8. The provided assay buffer, pH = 7.4, is recommended.
2. HRP stock solution (500X):
Add 100 µL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).
3. Xanthine Oxidase (XO) standard solution (1 U/mL)
Add 200 µL of Assay Buffer (Component B) into the vial of Xanthine Oxidase Standard (Component E).
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11304
Add 10 µL of 1 U/mL XO stanndard solution into 990 µL of Assay Buffer (Component B) to make 10 mU/mL XO standard solution (XO7). Perform 1:3 serial dilutions to get remaining serially diluted XO standards (XO6-XO1).
PREPARATION OF WORKING SOLUTION
Add 20 μL of Amplite™ Red stock solution (250X), 10 μL of HRP stock solution (500X), and 50 μL of Xanthine (100X, Component D) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.08 mL Xanthine Oxidase (XO) working solution. Protect from light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of XO standards and test samples in a solid black 96-well microplate.
BL | BL | TS | TS |
XO1 | XO1 | ... | ... |
XO2 | XO2 | ... | ... |
XO3 | XO3 | ||
XO4 | XO4 | ||
XO5 | XO5 | ||
XO6 | XO6 | ||
XO7 | XO7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
XO1 - XO7 | 50 µL | serial dilution (0.01 to 10 mU/mL) |
BL | 50 µL | Assay Buffer (Component B) |
TS | 50 µL | sample |
- Prepare XO standards (XO), 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.
- Add 50 µL of XO working solution into each well of the XO standards, blank control, and test samples to make the total XO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of XO working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction for 15 to 30 minutes at room temperature, protected from light.
- Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm), cut off = 570 nm.
Product Family
Images
Citations
Authors: Zhu, Li-Hua and Xu, Ying-Yin and Zhu, Li-ping and Zheng, Xian and Jiang, Cui-Hua and Liu, Jian-Jing and Zhang, Jian and Yin, Zhi-Qi
Journal: Journal of Functional Foods (2022): 105130
Authors: Singh, Sukhpal and Mahajan, Amita and Kaur, Jaspreet
Journal: Research Journal of Pharmacy and Technology (2020): 801--809
Authors: Ives, Annette and Nomura, Johji and Martinon, Fabio and Roger, Thierry and LeRoy, Didier and Miner, Jeffrey N and Simon, Gregoire and Busso, Nathalie and So, Alex and er, undefined
Journal: Nature communications (2015)
Authors: Ives, Annette and Nomura, Johji and Martinon, Fabio and Roger, Thierry and LeRoy, Didier and Miner, Jeffrey N and Simon, Gregoire and Busso, Nathalie and So, Alexander
Journal: Nature communications (2015): 1--11
References
Authors: Hanachi N, Charef N, Baghiani A, Khennouf S, Derradji Y, Boumerfeg S, Harzallah D, Arrar L.
Journal: Saudi Med J (2009): 1422
Authors: Hason S, Stepankova S, Kourilova A, Vetterl V, Lata J, Fojta M, Jelen F.
Journal: Anal Chem (2009): 4302
Authors: Metz S, Thiel W.
Journal: J Am Chem Soc (2009): 14885
Authors: Sousa C, Pereira DM, Valentao P, Ferreres F, Pereira JA, Seabra RM, Andrade PB.
Journal: J Agric Food Chem (2009): 2288
Authors: Sanchez-Cruz P, Alegria AE.
Journal: Chem Res Toxicol (2009): 818
Authors: Schmidt AP, Bohmer AE, Antunes C, Schallenberger C, Porciuncula LO, Elisabetsky E, Lara DR, Souza DO.
Journal: Br J Pharmacol (2009): 163
Authors: Mukojima K, Mishima S, Oda J, Homma H, Sasaki H, Ohta S, Yukioka T.
Journal: J Burn Care Res (2009): 335
Authors: Ernst ME, Fravel MA.
Journal: Clin Ther (2009): 2503
Authors: Tan WJ, Xu JC, Li L, Chen KL.
Journal: Nat Prod Res (2009): 393
Authors: Chen T, Guo ZP, Zhang YH, Gao Y.
Journal: Clin Rheumatol (2009): 1355
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