Amplite® Fluorimetric 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 fluorescence increase at Ex/Em = 540/590 nm

Important notes
To achieve the best results, it’s strongly recommended to use the black plates. 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 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) into a solid black 96-well microplate 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 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 fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off at 570 nm).