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Amplite® Fluorimetric L-Alanine Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare test samples along with serially diluted L-Alanine standards (50 µL)
- Add equal volume of L-Alanine working solution (50 µL)
- Incubate at 37°C for 30 minutes to 1 hour
- Monitor fluorescence intensity at Ex/Em = 540/590 nm
Important notes
To achieve the best result, it’s strongly recommended to use the black plates. Thaw kit components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. Quest Fluor™ L-Alanine Sensor stock solution (200X):
Add 55 µL of DMSO (Component E) into Quest Fluor™ L-Alanine Sensor (Component A) to make 200X Quest Fluor™ L-Alanine Sensor stock solution.
2. L-Alanine standard solution (1 mM):
Add 10 µL of 100 mM L-alanine (Component D) into 990 µL PBS (pH 7.0) to get 1 mM L-alanine solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13825
Add 100 µL of 1 mM L-Alanine standard solution into 900 µL PBS to make 100 µM L-alanine solution (AS7). Perform 1:2 serial dilutions to get serially diluted L-alanine standards (AS6 - AS1).
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 uL of 200X L-alanine sensor stock solution into the Enzyme Mix 1 bottle; mix well. Note: The working solution is not stable. Use promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of L-alanine standards and test samples in a solid black 96-well microplate. AS = L-Alanine standard (AS1 - AS7, 1.5 to 100 µM); BL = blank control; TS = test sample.
| BL | BL | TS | TS |
| AS1 | AS1 | ... | ... |
| AS2 | AS2 | ... | ... |
| AS3 | AS3 | ||
| AS4 | AS4 | ||
| AS5 | AS5 | ||
| AS6 | AS6 | ||
| AS7 | AS7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| AS1 - AS7 | 50 µL | Serial Dilution (1.5 to 100 µM) |
| BL | 50 µL | 1X PBS Buffer |
| TS | 50 | Test Sample |
- Prepare L-Alanine standards (AS), 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 L-Alanine working solution to each well of L-Alanine standard, blank control, and test samples to make the total L-Alanine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-Alanine working solution into each well instead, for a total volume of 50 µL/well. Note: Run the L-alanine assay at pH 6.5 to 7.0.
- Incubate the reaction mixture at 37°C for 30 minutes to 1 hour.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (cut off: 570 nm).
Citations
Authors: Lyu, Fengzhi and Zhang, Tianyu and Yang, Dong and Gui, Meng and Wang, Yongtao and Liao, Xiaojun and Rao, Lei
Journal: bioRxiv (2024): 2024--03
Authors: Skibbe, K and Brethack, AK and S{\"u}nderhauf, A and Ragab, M and Raschdorf, A and Hicken, M and Schlichting, H and Preira, J and Brandt, J and Castven, D and others,
Journal: (2021)
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Journal: J Phys Chem B (2011): 15339
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Journal: Guang Pu Xue Yu Guang Pu Fen Xi (2010): 2620
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