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Amplite® Fluorimetric Aspartate Aminotransferase (AST) Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare AST working solution (50 µL)
- Add AST standards or test samples (50 µL)
- Incubate at 37 °C for 20 - 30 min or RT for 60 min
- Monitor fluorescence increase at Ex/Em = 540/590 nm
Important notes
Thaw one bottle each of Component A and B at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. AST standard solution (100 U/mL):
Add 100 µL DPBS Buffer to AST Positive Control (Component D) to make 100 U/mL AST standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13800
Add 3 µL of 100 U/mL AST standard solution into 997 µL DPBS buffer with 0.1% BSA to generate 300 mU/mL AST standard solution (AST7). Take 300 µL of 300 mU/mL AST standard solution to perform 1:3 serial dilutions to get serial dilutions of AST standard (AST6 - AST1). Note: The AST standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.
PREPARATION OF WORKING SOLUTION
1. NAD solution (100X):
Add 100 µL of ddH2O into the vial of NAD (Component C).
2. AST Enzyme Mixture solution:
Add 10 mL of AST Assay Buffer (Component B) into the bottle of AST Enzyme Mixture (Component A), and mix well.
3. AST working solution:
Add the whole vial of 100X NAD solution (from 1) into the AST Enzyme Mixture solution (from 2) to have AST working solution. Note: This AST working solution is enough for two 96-well plates. It is unstable at room temperature, and should be used promptly within 2 hours. Alternatively, one can make a 50X of AST Enzyme Mixture stock solution by adding 200 µL of H2O into the bottle of Component A, and then prepare the AST working solution by mixing the stock solution with assay buffer (Component B) and 100X NAD solution proportionally.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of AST standards and test samples in a solid black 96-well microplate. AST= AST Standards (AST1 - AST7, 0.3 to 300 mU/mL), BL=Blank Control, TS=Test Samples.
| BL | BL | TS | TS |
| AST1 | AST1 | ... | ... |
| AST2 | AST2 | ... | ... |
| AST3 | AST3 | ||
| AST4 | AST4 | ||
| AST5 | AST5 | ||
| AST6 | AST6 | ||
| AST7 | AST7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| AST1-AST7 | 50 µL | Serial Dilution (0.3 to 300 mU/mL) |
| BL | 50 µL | DPBS with 0.1% BSA |
| TS | 50 µL | Sample |
- Prepare AST standards (AST), 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 AST working solution to each well of AST standard, blank control, and test samples to make the total AST assay volume of 100 µL/well. For a 384-well plate, add 25 µL of AST working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at 37°C for 20 - 30 minutes or room temperature for 60 minutes, protected from light. Note: The background of Blank Control increases with time and temperature.
- 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).
Citations
Authors: Wei, Ching-Ting and Wang, Yu-Wen and Wu, Yu-Chiuan and Lin, Li-Wei and Chen, Chia-Chi and Chen, Chun-Yin and Kuo, Shyh-Ming
Journal: Pharmaceutics (2022): 1108
Authors: Weinhage, Toni and Wirth, Timo and Sch{\"u}tz, Paula and Becker, Philipp and Lueken, Aloys and Skryabin, Boris V and Wittkowski, Helmut and Foell, Dirk
Journal: Frontiers in immunology (2020): 1157
Authors: Wu, Yi Jhen and Wu, Yu Chiuan and Chen, I-Fen and Wu, Yi-Lung and Chuang, Chin Wen and Huang, Han Hsiang and Kuo, Shyh Ming
Journal: Molecules (2018): 726
Authors: Tsai, Meng-Tsz and Katagiri, Naohiro and Ohbayashi, Norihiko and Iwasaki, Kenichi and Ohkohchi, Nobuhiro and Ding, Shih-Torng and Kanaho, Yasunori and Funakoshi, Yuji
Journal: Scientific reports (2017): 1--12
Authors: Chang, Siou Han and Huang, Han Hsiang and Kang, Pei Leun and Wu, Yu Chian and Chang, Ming-Huang and Kuo, Shyh Ming
Journal: Acta Biomaterialia (2017)
References
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Journal: Skin Res Technol (2006): 155
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Journal: Auris Nasus Larynx. (2006)
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Journal: Clin Experiment Ophthalmol (2006): 33
Authors: Shmyreva VF, Petrov S, Novikov IA, Zueva Iu S.
Journal: Vestn Oftalmol (2005): 3
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