Amplite® Colorimetric Hexokinase Assay Kit

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

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

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

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 of NADPH standard (Component G) to make a 2 mM (2 nmol/µL) NADPH stock solution. Store the solution at -80°C. Avoid repeated freeze/thaw cycles.

1. To reconstitute the Hexokinase Positive Control (Component F), add 50 µL of ddH2O to prepare a 100 µg/mL stock solution. Mix well by pipetting and store at –20 °C.

   Note: Must be used within 2 months of reconstitution.

1. To prepare a 100X HK Substrate stock solution, add 100 µL of ddH2O to the vial containing the HK Substrate (Component D). After mixing, store the solution at -20°C. Avoid repeated freezing and thawing.

1. Add 100 µL of ddH2O to the vial containing HK Coenzyme (Component E) to create a 100X stock solution. Store this solution at -20°C, and avoid repeated freeze/thaw cycles.

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

2. Transfer 5 mL of the above-prepared buffer mixture into the HK Enzyme Mix bottle (Component C) and mix thoroughly.

3. Add 50 µL of the HK Substrate stock solution to the same bottle and mix well.

4. Add 50 µL of the HK Coenzyme stock solution to the same bottle and mix well.

   Note: This HK working solution should be freshly prepared before each experiment and protected from light. A 5.0 mL solution is enough for 100 tests. Please prepare the necessary amount of HK working solution based on this proportion.

   Note: Alternatively, one can make a 50X HK Enzyme Mix stock solution by adding 100 μL of ddH2O into the bottle of HK Enzyme Mix (Component C) and then prepare the HK working solution by mixing the HK Enzyme Mix stock solution with other components listed above in the 'HK Working Solution' proportionally.

    

1. Prepare one or more HK positive control samples along with the test sample. The recommended concentration for the HK positive control is between 0.25 and 0.025 μg/ml in PBS.

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

Table 2. Reagent composition for each well.

1. Prepare NADPH standards (STD1-7), blank controls (BL), HK Positive Control, and test samples (TS) as outlined 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 HK Working Solution to each well containing the NADPH standard, blank control, HK Positive Control, and test samples. For a 384-well plate, add 25 µL of HKNE 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.