Amplite® Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*

Protocol summary

1. Glutamate Oxidase standards or test samples (50 µL)
2. Add Glutamate Oxidase 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 to room temperature before starting the experiment.

1. Amplite™ Red stock solution (250X):
Add 40 µL of DMSO (Component F) into the vial of Amplite™ Red (Component A). The stock solution should be used promptly. Note: The Amplite™ Red 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 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 (400X):
Add 200 µL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).

3. Glutamic Acid stock solution (400X):
Add 1.0 mL of ddH₂O into the vial of Glutamic Acid (Component D) to make 400X glutamic acid stock solution.

4. Glutamate Oxidase (GO) standard solution (150 mU/mL):
Add 100 µL of Assay Buffer (Component B) into the vial of Glutamate Oxidase Standard (lyophilized, Component E) to make 150 mU/mL Glutamate Oxidase (GO) standard solution.

Add 20 μL of Amplite™ Red stock solution (250X), 12.5 μL of HRP stock solution (400X) and 12.5 μL of Glutamic Acid stock solution (400X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.07 mL Glutamate Oxidase working solution(GO working solution). Protect from light.

Table 1. Layout of GO standards and test samples in a solid black 96-well microplate. GO= glutamate oxidase standards (GO1 - GO7, 0.01 to 10 mU/mL), BL=blank control, TS=test samples.

Table 2. Reagent composition for each well. Higher concentrations of GO may cause reduced fluorescence signal due to the over oxidation of Amplite™ Red (to a non-fluorescent product).

1. Prepare GO standards (GO), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of GO working solution to each well of GO standard, blank control, and test samples to make the total GO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of GO 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 intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm), cutoff = 570 nm. Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading.