Amplite® Fluorimetric Glycerol Assay Kit

Protocol summary

1. Prepare Glycerol working solution (50 µL)
2. Add Glycerol standards or test samples (50 µL)
3. Incubate at room temperature for 10 - 30 minutes
4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
To achieve the best results, it's strongly recommended to use the black plates. Thaw one vial of each kit component at room temperature before starting the experiment.

1. Amplite™ HRP Substrate stock solution (200X):
Add 50 µL of DMSO (Component E) into the vial of Amplite™ HRP Substrate (Component A) to make 200X Amplite™ HRP Substrate stock solution. Protect from light.

2. Glycerol standard solution (1 mg/mL):
Add 1 mL of ddH2O or 1x PBS buffer into the vial of Glycerol Standard (Component D) to make 1 mg/mL Glycerol standard solution.

1. Add 5 mL of Assay Buffer (Component C) into a bottle of Enzyme Mix (Component B), and mix welll.

2. Add 25 µL of 200X Amplite™ HRP Substrate stock solution into the bottle of Component B+C, and mix well to make Glycerol working solution. Note: This Glycerol working solution is enough for one 96-well plate. It is not stable, use it promptly.

Table 1. Layout of Glycerol standards and test samples in a solid black 96-well microplate. GS= Glycerol Standards (GS1 - GS7, 0.01 to 10 µg/mL), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well

1. Prepare Glycerol standards (GS), 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 Glycerol working solution to each well of Glycerol standard, blank control, and test samples to make the total Glycerol assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Glycerol working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).