Amplite® Colorimetric Glycerol 3-Phosphate (G3P) Assay Kit

1. Prepare Glycerol 3-Phosphate standards or test samples (50 µL)
2. Add Glycerol 3-Phosphate working solution (50 µL)
3. Incubate at RT for 30 min to 1 hour
4. Monitor absorbance increase at OD of 575 nm

Thaw all the kit components 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™ Red substrate (Component A) to make a 200X stock solution. Avoid exposure to light.
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 250 µL of ddH₂O (Component B) into the vial of G3P Standard (Component D) to make 10 mM G3P standard solution.

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

Add 25 µL of Amplite™ Red substrate stock solution (200X) into the same bottle of Enzyme Mix (Components B) to make the G3P working solution (final bottle should contain Components A, B and C). Note: This working solution is not stable, use it promptly and avoid direct exposure to light.

Table 1. Layout of G3P standards and test samples in a clear bottom 96-well microplate. CS = G3P standard (CS1 - CS7, 3.12 to 200 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

1. Prepare G3P standards (CS), blank controls (BL), and test samples (TS) into a 96-well clear bottom/black wall microplate 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. Note: Treat the cells or tissues as desired.
2. Add 50 µL of G3P working solution to each well of G3P standard, blank control, and test samples to make total G3P assay volume of 100 µL/well. For a 384-well plate, add 25 µL of G3P working solution into each well instead, for a total volume of 50 µL/well.
3. Incubate the reaction at room temperature for 30 min to 1 hour, protected from light.
4. Monitor the absorbance increase with an absorbance microplate reader at 575 nm (or ratio of 570 nm/610 nm).