Calculator
Amplite® Colorimetric 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 37ºC for 30 - 120 minutes
- Monitor absorbance increase at the absrobance ratio of A570nm /A610nm
Important notes
Thaw the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. AST standard solution (100 U/ml):
Add 100 µL DPBS buffer into the vial of 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/13801
Add 5 μL of 100 U/mL AST standard solution into 997 μL DPBS buffer with 0.1% BSA to generate 500 mU/mL AST standard solution (AST7). Take 500 mU/mL AST standard solution (AST7) and perform 1:2 serial dilutions in DPBS buffer with 0.1% BSA to get serial diluted AST standards (AST6 - AST1).
PREPARATION OF WORKING SOLUTION
1. Add 100 μL of ddH2O into the vial of NAD (Component C) to have 100X NAD solution.
2. Add 10 mL of AST Assay Buffer (Component B) into the bottle of AST Enzyme Mixture (Component A), and mix well. Then, add the whole vial of 100X NAD solution into the AST Enzyme Mixture solution to make 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. Avoid exposure to light. Note: Alternatively, one can make a 50X of AST Enzyme Mixture stock solution by adding 200 μL of H2O into the bottle of AST Enzyme Mixture (Component A), and then prepare the AST working solution by mix 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 clear bottom 96-well microplate. AST= AST Standards (AST1 - AST7, 7.8 to 500 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. Note: The AST standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.
| Well | Volume | Reagent |
| AST1 - AST7 | 50 µL | Serial Dilutions (7.8 to 500 mU/mL) |
| BL | 50 µL | DPBS with 0.1% BSA |
| TS | 50 µL | test 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 37ºC for 30 - 120 minutes, protected from light. Note: The background of Blank Control increases with time and temperature.
- Monitor the absorbance increase with an absorbance plate reader at the absorbance ratio of A570nm /A610nm.
AST assay calculator
This tool will generate a linear regression model for any given experimental data set with which to determine an unknown AST concentration for the AST Assay.
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: Shiu, Li-Yen and Huang, Han Hsiang and Chen, Chun Yin and Cheng, Hsia-Ying and Chen, Chih I and Kuo, Shyh Ming
Journal: Bioscience Reports (2020)
Authors: Liu, Pengfei and de la Vega, Montserrat Rojo and Dodson, Matthew and Yue, Fei and Shi, Boyun and Fang, Deyu and Chapman, Eli and Liu, Leyuan and Zhang, Donna D
Journal: Hepatology (2019)
Authors: Hassan, Faizule and Lossie, Sarah L and Kasik, Ellen P and Channon, Audrey M and Ni, Shuisong and Kennedy, Michael A
Journal: PloS one (2018): e0192882
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
Authors: Mehta S., undefined
Journal: Indian J Ophthalmol (2006): 273
Authors: O'Goshi K, Serup J.
Journal: Skin Res Technol (2006): 155
Authors: Anari S, Waldron M, Carrie S.
Journal: Auris Nasus Larynx. (2006)
Authors: Kwan AS, Barry C, McAllister IL, Constable I.
Journal: Clin Experiment Ophthalmol (2006): 33
Authors: Shmyreva VF, Petrov S, Novikov IA, Zueva Iu S.
Journal: Vestn Oftalmol (2005): 3
Safety data sheet