Amplite® Fluorimetric Xanthine Assay Kit

Protocol summary

1. Prepare xanthine standards or test samples (50 µL)
2. Add xanthine working solution (50 µL)
3. Incubate at room temperature for 30 - 60 min
4. Read fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. Amplite™ Red substrate stock solution (250X):
Add 40 µL of DMSO (Component F) into the vial of Amplite™ Red substrate (Component A). Note: 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 assay should be performed at pH 7 - 8 (pH 7.4 is recommended) as Amplite™ Red is unstable at pH > 8.5.

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) stock solution (100X):
Add 100 µL of Assay Buffer (Component B) into the vial of Xanthine Oxidase (Component E) to make Xanthine Oxidase (XO) stock solution (100X).

Add 20 μL of Amplite™ Red Substrate stock solution (250X), 10 μL of HRP stock solution (500X), and 50 μL of Xanthine Oxidase stock solution (100X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.08 mL. Note: Avoid direct exposure to light and use promptly.

Table 1. Layout of Xanthine standards and test samples in a black wall/solid bottom 96-well microplate. X = xanthine standard (X1 - X7, 0.137 to 100 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

1. Prepare xanthine standards (X), blank controls (BL), and test samples (TS) 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.
   
   
2. Add 50 µL of xanthine working solution into each well of the xanthine standards, blank control, and test samples to make the total xanthine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.
   
   
4. Monitor the fluorescence increase with with a fluorescence plate reader at Excitation = 530 - 570 nm (optimal at 540 nm), Emission = 590 - 600 nm (optimal at 590 nm), cutoff = 570 nm.