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

1. Prepare G3P standards or test samples (50 µL)
2. Add G3P working solution (50 µL)
3. Incubate at RT for 10 - 30 min
4. Read fluorescence intensity at Ex/Em = 540/590 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.

Add 250 µL of ddH₂O 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 bottle of Components B and C to make the G3P working solution (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 solid black 96-well microplate. CS = G3P standard (CS1 - CS7, 0.1 to 100 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each cell.

1. Prepare G3P standards (CS), blank controls (BL), and test samples (TS) into a 96-well black 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.
2. Add 50 µL of G3P working solution into each well of G3P standard, blank control, and test samples to make the total G3P assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
3. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (cut off at 570 nm).