Amplite® Colorimetric Beta-Hydroxybutyrate (Ketone Body) Assay Kit

Protocol summary

1. Prepare β-HB working solution (50 µL)
2. Add β-HB standards or test samples (50 µL)
3. Incubate at room temperature for 10 - 30 min
4. Monitor Absorbance increase at OD ratio of 570/610 nm

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

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

2. β-HB standard solution (100 mM):
Add 1 mL of H₂O or 1X PBS buffer into the vial of β-HB standard (Component D) to make 100 mM β-HB standard solution.

1. Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Mix (Component A).

2. Add 50 µL of 100X NAD stock solution into the bottle of Component A+B, and mix well to make β-HB working solution (Component A+B+C). Note: This β-HB working solution is not stable, use it promptly and avoid direct exposure to light.

Table 1. Layout of β-HB standards and test samples in a clear bottom 96-well microplate. HB = β-HB standard (HB1 - HB7, 1 to 1000 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

1. Prepare β-HB standards (HB), blank control (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 β-HB working solution to each well of β-HB standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of β-HB working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.
   
   
4. Monitor the absorbance increase with an absorbance plate reader at OD ratio of 570/610 nm.