Amplite® Colorimetric Beta-Hydroxybutyrate (Ketone Body) Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Platform
Absorbance microplate reader
Absorbance | 570/610 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
Protocol summary
- 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
Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
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.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13830
Add 10 µL of 100 mM β-HB standard stock solution into 990 µL 1X PBS buffer to generate 1000 µM β-HB standard solution (HB7). Take the 1000 µM β-HB standard solution (HB7) and perform 1:3 serial dilutions in PBS to get serially diluted β-HB standards (HB6 - HB1). Note: Diluted β-HB standard solution is unstable, and should be used within 4 hours.
PREPARATION OF WORKING 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.
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.
Images
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 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
Authors: Morales, Ana
Journal: (2020)
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: Bonartsev AP, Yakovlev SG, Zharkova, II, Boskhomdzhiev AP, Bagrov DV, Myshkina VL, Makhina TK, Kharitonova EP, Samsonova OV, Feofanov AV, Voinova VV, Zernov AL, Efremov YM, Bonartseva GA, Shaitan KV, Kirpichnikov MP.
Journal: BMC Biochem (2013): 12
Authors: Cheng B, Lu H, Bai B, Chen J.
Journal: Neurochem Int (2013): 620
Authors: Lim J, Chong MS, Teo EY, Chen GQ, Chan JK, Teoh SH.
Journal: J Biomed Mater Res B Appl Biomater (2013): 752
Authors: Lomas AJ, Webb WR, Han J, Chen GQ, Sun X, Zhang Z, El Haj AJ, Forsyth NR.
Journal: Tissue Eng Part C Methods (2013): 577
Authors: Vogensen SB, Marek A, Bay T, Wellendorph P, Kehler J, Bundgaard C, Frolund B, Pedersen MH, Clausen RP.
Journal: J Med Chem (2013): 8201
Authors: Zheng J, Li D, Yuan L, Liu X, Chen H.
Journal: ACS Appl Mater Interfaces (2013): 5882
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