Amplite® Fluorimetric Coenzyme A Quantitation Kit *Green Fluorescence*

Protocol summary

1. Prepare CoA working solution (50 µL)
2. Add CoA standards or test samples (50 µL)
3. Incubate at RT for 10 minutes - 1 hour
4. Monitor the fluorescence increase at Ex/Em = 490/520 nm

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

1. CoA standard solution (1 mM):
Add 200 µL of ddH₂O into the CoA standard vial (Component C) to make 1 mM (1 nmol/µL) stock solution. Note: It is highly recommended to use the ddH₂O that has been sparged with nitrogen to remove oxygen for preparing coenzyme A standard solution. The aqueous solution is not stable and will degrade rapidly. It should be stored at 2 - 8 °C and used within the day.

2. CoA Green™ stock solution (100X):
Add 100 µL of DMSO (Component D) into the vial of CoA Green™ (Component A) to make 100X stock solution.

Add 50 μL of CoA Green™ stock solution (100X) into 5 mL of Assay Buffer (Component B) and mix well.

Table 1. Layout of CoA standards and test samples in a solid black 96-well microplate. CoA = Coenzyme A standard (CoA1 - CoA7, 0.01 to 10 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

1. Prepare coenzyme A standards (CoA), blank controls (BL), and test samples (TS) according ot 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 CoA working solution to each well of the CoA standard, blank control, and test sample to make the total CoA assay volume of 100 µL/well. For a 384-well plate, add 25 µL of CoA working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 10 minutes to 1 hour, protected from light.
   
   
4. Monitor the fluorescence increase at Ex/Em = 490/520 nm with a fluorescence plate reader.