Amplite® Colorimetric Phosphofructokinase (PFK) Activity Assay Kit

Thaw all the kit components at room temperature before starting the experiment.

1. Prepare the test samples, and the serially diluted NADH standards (50 μL).

2. Add the PFK working solution (50 μL).

3. Incubate for 10-30 minutes at room temperature.

4. Measure the absorbance at 450 nm.

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

1. Add 200 µL of PBS buffer to the vial containing the NADH standard (Component F) to prepare a 2 mM (2 nmol/µL) NADH stock solution.

   Note: Store the solution at -80°C. Avoid repeated freeze/thaw cycles.

1. Reconstitute the Phosphofructokinase (PFK) Positive Control (Component G) by adding 100 µL of ddH2O, and mix well by pipetting.

   Note: Store at -20°C, and use within 2 months of reconstitution.

1. Add 100 µL of ddH2O to the vial of ATP (Component E) to create a 100X ATP stock solution.

   Note: Store this solution at -20°C and avoid repeated freeze/thaw cycles.

1. Add 100 µL of ddH2O to the vial of PFK Developer (Component D) to create a 100X PFK Developer stock solution.

   Note: Store this stock solution at -20°C. Avoid repeated freeze/thaw cycles.

1. Add 1.0 mL of PFK Probe (Component A) to 4 mL PFK Assay Buffer (Component B), and mix well.

2. Transfer the 5 mL buffer mixture from above to the PFK Enzyme Mix bottle, and mix well.

3. Add 50 µL of the ATP stock solution to the same bottle, and mix well.

4. Add 50 µL of the PFK Developer stock solution to the same bottle, and mix well.

   Note: Prepare the PFK working solution fresh before each experiment and protect it from light. A 5 mL preparation is sufficient for 100 tests. Adjust the volume proportionally based on the number of tests you plan to conduct.

   Note: Alternatively, one can prepare a 50X stock solution of PFK Enzyme Mix by adding 100 μL of ddH2O to the bottle of PFK Enzyme Mix (Component C) and mix thoroughly to dissolve the enzyme completely. Next, to prepare the PFK working solution, combine the 50X stock solution with the other components listed in the 'PFK Working Solution' section above, following the specified proportions.

Table 1. Layout of NADH standards and test samples in a 96-well solid black microplate. (STD = NADH Standards (STD1-STD7, 3.125-200 µM), BL = Blank Control, TS = Test Samples)

Table 2. Reagent composition for each well.

1. Prepare the NADH standards (STD1-7), blank controls (BL), PFK Positive Control, and test samples (TS) as outlined in Tables 1 and 2. When using a 384-well plate, add 25 µL of reagent to each well instead of the standard 50 µL.

2. Add 50 µL of PFK Working Solution to each well containing the NADH standard, blank control, PFK Positive Control, and test samples. If you are using a 384-well plate, add 25 µL of PFK Working Solution to each well instead.

3. Incubate at room temperature for 10-30 minutes, protected from light.

4. Monitor the absorbance intensity with an absorbance microplate reader at 450 nm.