Amplite® Colorimetric Beta-Hydroxybutyrate (Ketone Body) Assay Kit
Example protocol
AT A GLANCE
- Prepare β-HB working solution (50 µL)
- Add β-HB standards or test samples (50 µL)
- Incubate at room temperature for 10 - 30 min
- Monitor Absorbance increase at OD ratio of 570/610 nm
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTIONS
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
Add 100 µL of H2O into the vial of NAD (Component C) to make 100X NAD stock solution.
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.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/13830
PREPARATION OF WORKING SOLUTION
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).
Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Mix (Component A).
Note: This β-HB working solution is not stable, use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
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.
BL | BL | TS | TS |
HB1 | HB1 | ... | ... |
HB2 | HB2 | ... | ... |
HB3 | HB3 | ||
HB4 | HB4 | ||
HB5 | HB5 | ||
HB6 | HB6 | ||
HB7 | HB7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
HB1-HB7 | 50 µL | Serial Dilution (1 to 1000 µM) |
BL | 50 µL | 1X PBS Buffer |
TS | 50 µL | Test Sample |
- 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.
- 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.
- Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.
- Monitor the absorbance increase with an absorbance plate reader at OD ratio of 570/610 nm.
Citations
Authors: Autterson, Gillian and Han, John Yeong Se and Philp, Nancy and Miller, Jason Matthew Lewis
Journal: Investigative Ophthalmology \& Visual Science (2023): 4466--4466
Authors: Gumus, Hikmet and Ilgin, Rabia and Koc, Basar and Yuksel, Oguz and Kizildag, Servet and Guvendi, Guven and Karakilic, Asli and Kandis, Sevim and Hosgorler, Ferda and Ates, Mehmet and others,
Journal: Neuroscience Letters (2022): 136443
Authors: Hirata, Yoshiki and Shimazaki, Sayaka and Suzuki, Sae and Henmi, Yuka and Komiyama, Hiromu and Kuwayama, Takehito and Iwata, Hisataka and Karasawa, Tadayoshi and Takahashi, Masafumi and Takahashi, Hironori and others,
Journal: Journal of Reproductive Immunology (2021): 103433
Authors: Morales, Ana
Journal: (2020)
Authors: Morales, Ana and Frei, Barbara and Leung, Casey and Titman, Rodger and Whelan, Shannon and Benowitz-Fredericks, Z Morgan and Elliott, Kyle H
Journal: Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology (2020): 110594
References
Authors: Wang W, Lan P.
Journal: J Biomater Sci Polym Ed (2014): 2094
Authors: Zharkova, II, Staroverova OV, Voinova VV, Andreeva NV, Shushckevich AM, Sklyanchuk ED, Kuzmicheva GM, Bespalova AE, Akulina EA, Shaitan KV, Olkhov AA.
Journal: Biomed Khim (2014): 553
Authors: Johansson J, Gronbladh A, Hallberg M.
Journal: Behav Brain Res (2014): 164
Authors: Wei T, Tian W, Liu F, Xie G.
Journal: Biochem Biophys Res Commun (2014): 666
Authors: Zheng J, Li D, Yuan L, Liu X, Chen H.
Journal: ACS Appl Mater Interfaces (2013): 5882