Amplite® Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*
![Xanthine oxidase dose response was measured with Amplite® Fluorimetric Xanthine Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-xanthine-oxidase-assay-kit-red-fluorescence%2Fgraph-for-amplite-fluorimetric-xanthine-oxidase-assay-kit-red-fluorescence_k40t5.webp&w=640&q=75)
![Xanthine oxidase dose response was measured with Amplite® Fluorimetric Xanthine Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-xanthine-oxidase-assay-kit-red-fluorescence%2Fgraph-for-amplite-fluorimetric-xanthine-oxidase-assay-kit-red-fluorescence_k40t5.webp&w=640&q=75)
![Xanthine oxidase dose response was measured with Amplite® Fluorimetric Xanthine Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-xanthine-oxidase-assay-kit-red-fluorescence%2Fgraph-for-amplite-fluorimetric-xanthine-oxidase-assay-kit-red-fluorescence_k40t5.webp&w=128&q=25)
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.
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