Amplite® Colorimetric α-Ketoglutarate Quantitation 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 | 12171501 |
Overview | ![]() ![]() |
Platform
Absorbance microplate reader
Absorbance | 575 nm |
Recommended plate | Clear bottom |
Instrument specification(s) | Path check |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare test samples along with serially diluted α-ketoglutarate standards (50 µL)
- Add equal volume of α-Ketoglutarate working solution (50 µL)
- Incubate at 37°C for 60 - 90 minutes
- Monitor absorbance intensity at 570 nm
Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Amplite™ Red stock solution (200X):
Add 50 µL of DMSO (Component E) into the vial of Amplite™ Red (Component A) to make 200X stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10085
Add 10 µL of 10 mM α-Ketoglutarate Standard (Component D) into 990 µL of PBS to get 100 µM α-ketoglutarate standard solution (AKG7). Then perform 1:2 serial dilutions to get serially diluted α-ketoglutarate standards (AKG6 - AKG1).
PREPARATION OF WORKING SOLUTION
1. Add 5 mL Assay Buffer (Component C) into one Enzyme Mix 1 bottle (Component B1) and mix well.
2. Add 100 μL of ddH2O into one Enzyme Mix 2 vial (Component B2) and mix well.
3. Transfer entire vial (100 μL) of Enzyme Mix 2 and 25 µL of 200X Amplite™ Red stock solution into the vial of Enzyme Mix 1 and mix well to make α-Ketoglutarate working solution. Note: The 5 mL α-Ketoglutarate working solution is enough for one 96-well plate. It is not stable, use it promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of α-ketoglutarate standards and test samples in a 96-well clear bottom microplate. AKG= α-Ketoglutarate Standard (AKG1 - AKG7, 1.563 to 100 µM), BL=Blank Control, TS=Test Sample.
BL | BL | TS | TS |
AKG1 | AKG1 | ... | ... |
AKG2 | AKG2 | ... | ... |
AKG3 | AKG3 | ||
AKG4 | AKG4 | ||
AKG5 | AKG5 | ||
AKG6 | AKG6 | ||
AKG7 | AKG7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
AKG1 - AKG7 | 50 µL | Serial Dilutions (1.563 to 100 µM) |
BL | 50 µL | Assay Buffer (Component C) |
TS | 50 µL | test sample |
- Prepare α-ketoglutarate standards (AKG), blank controls (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 α-Ketoglutarate working solution to each well of α-ketoglutarate standard, blank control, and test samples to make the total α-ketoglutarate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of α-Ketoglutarate working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction mixture at 37°C for 60 - 90 minutes.
- Monitor the absorbance increase with an absorbance plate reader with path check on at OD of 570 nm.
Images
Citations
Authors: Jia, Yinzhao and Yin, Chuanzheng and Ke, Wenbo and Liu, Jing and Guo, Bing and Wang, Xiaofei and Zhao, Peng and Hu, Shaobo and Zhang, Chen and Li, Xuan and others,
Journal: Science of The Total Environment (2023): 163069
References
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Journal: Neuron (2017): 115
Authors: Bayliak MM, Lylyk MP, Shmihel HV, Sorochynska OM, Semchyshyn OI, Storey JM, Storey KB, Lushchak VI.
Journal: Comp Biochem Physiol A Mol Integr Physiol (2017): 28
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Journal: J Am Chem Soc (2016): 5110
Authors: Fiehn O, Showalter MR, Schaner-Tooley CE.
Journal: Elife (2016)
Authors: Chen H, Denton TT, Xu H, Calingasan N, Beal MF, Gibson GE.
Journal: J Neurochem (2016): 823
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