Amplite® Colorimetric Glycerol Assay Kit

1. Prepare glycerol standards or test samples (50 µL)
2. Add glycerol working solution (50 µL)
3. Incubate at room temperature for 30 min to 1 hour
4. Monitor OD at 575 nm

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.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 50 µL of DMSO (Component E) into the vial of Amplite™ HRP substrate (Component A) to make 200X stock solution. 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.

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.

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

Add 25 µL of Amplite™ HRP substrate stock solution into the bottle of Component B + C and mix them well to make glycerol working solution (Component A + B + C). Note: This 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 clear bottom 96-well microplate. G= Glycerol Standards (G1 - G7, 0.156 to 10 µg/mL), BL=Blank Control, TS=Test Samples.

Table 2. Reagent composition for each well.

1. Prepare glycerol standards (G), 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 30 minutes to 1 hour, protected from light.
4. Monitor the absorbance increase with an absorbance plate reader with path check on at OD of 575 nm.