Amplite® Fluorimetric Glucose-6-Phosphate Assay Kit

Protocol summary

1. Prepare G6P working solution (50 µL)
2. Add G6P standards or test samples (50 µL)
3. Incubate at room temperature for 30 minutes - 2 hours
4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

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

1. NADP stock solution (100X):
Add 100 µL of H₂O into the vial of NADP (Component C) to make 100X NADP stock solution.

2. G6P standard solution (100 mM):
Add 100 µL of H₂O or 1x PBS buffer into the vial of G6P Standard (Component D) to make 100 mM G6P standard solution.

1. Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Probe (Component A), and mix well.

2. Add 50 µL of 100X NADP stock solution into the bottle of Component A+B, and mix well to make G6P working solution. Note: This G6P working solution is enough for one 96-well plate.

Table 1. Layout of G6P standards and test samples in a solid black 96-well microplate. G6P=G6P Standards (G6P1 - G6P7, 0.14 to 100 µM), BL=Blank Control, TS=Test Samples.

Table 2. Reagent composition for each well.

1. Prepare G6P standards (G6P), 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 G6P working solution to each well of G6P standard, blank control, and test samples to make the total G6P assay volume of 100 µL/well. For a 384-well plate, add 25 µL of G6P 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 2 hours, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, Cutoff =  570nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by absorbance microplate reader at the ratio of A_575nm/A_605nm. The absorption detection has lower sensitivity compared to fluorescence reading.