Amplite® Fluorimetric Alanine Aminotransferase 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 | ![]() ![]() |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare ALT working solution (50 µL)
- Add ALT standards or test samples (50 µL)
- Incubate at 37°C for 30 min to 60 min
- Monitor fluorescence increase at Ex/Em = 540/590 nm
Important notes
Thaw one bottle Component A and B at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. ALT standard solution:
Add 100 µL DPBS into the vial of ALT Positive Control (Component D) to make 100 U/mL ALT stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13802
Add 10 µL of 100 U/mL ALT standard solution into 990 µL DPBS buffer with 0.1% BSA to generate 1000 mU/mL ALT standard solution (ALT7). Take the 1000 mU/mL ALT standard solution and perform 1:3 serial dilutions to get serial dilutions of ALT standard (ALT6 - ALT1).
PREPARATION OF WORKING SOLUTION
1. NAD solution (100X):
Add 100 µL of ddH2O into the vial of NAD (Component C).
2. ALT Enzyme Mixture solution:
Add 10 mL of ALT Assay Buffer (Component B) into the bottle of ALT Enzyme Mixture (Component A), and mix well.
3. ALT working solution:
Add the whole vial of 100X NAD solution (from 1) into the ALT Enzyme Mixture solution (from 2) to have ALT working solution.
Note: This ALT working solution is enough for two 96-well plates. It is unstable at room temperature, and should be used promptly within 2 hours and avoid exposure to light. Alternatively, one can make a 50X of ALT Enzyme Mixture stock solution by adding 200 μL of H2O into the bottle of Component A, and then prepare the ALT working solution by mixing the stock solution with assay buffer (Component B) and 100x NAD solution proportionally.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of ALT standards and test samples in a solid black 96-well microplate. ALT = ALT standard (ALT1 - ALT7, 1 to 1000 mU/mL); BL = blank control; TS = test sample.
BL | BL | TS | TS |
ALT1 | ALT1 | ... | ... |
ALT2 | ALT2 | ... | ... |
ALT3 | ALT3 | ||
ALT4 | ALT4 | ||
ALT5 | ALT5 | ||
ALT6 | ALT6 | ||
ALT7 | ALT7 |
Table 2. Layout of ALT standards and test samples in a solid black 96-well microplate.
Well | Volume | Reagent |
ALT1 - ALT7 | 50 µL | Serial Dilution (1 to 1000 mU/mL) |
BL | 50 µL | DPBS with 0.1% BSA |
TS | 50 µL | Test Sample |
- Prepare ALT standards (ALT), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according the the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Dilute the test samples to the appropriate concentration range (indicated by the ALT standards) in DPBS buffer with 0.1% BSA if needed.
- Add 50 µL of ALT working solution into each well of ALT standard, blank control, and test samples to make the total ALT assay volume of 100 µL/well. For a 384-well microplate, add 25 µL of ALT working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at 37°C for 30 min to 60 minutes, protected from light. Note: The background of Blank Control will increase with time.
- 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).
Images
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
References
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Application notes
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