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Amplite® Fluorimetric L-Aspartate (Aspartic Acid) Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare test samples along with diluted aspartate standards (50 µL)
- Add equal volume of working solution (50 µL)
- Incubate at 37°C for 20 - 30 minutes
- Monitor fluorescence intensity at Ex/Em = 540/590 nm
Important notes
Thaw kit components at room temperature before use. To achieve the best results, it’s recommended to use the black plates.
PREPARATION OF STOCK SOLUTION
1. Aspartate standard solution (10 mM):
Add 100 uL of ddH2O into Aspartate Standard vial (Component E) to make 10 mM aspartate standard solution.
2. Amplite™ Red substrate stock solution (200X):
Add 50 µL of DMSO (Component F) into Amplite™ Red substrate (Component A) to make 200X Amplite™ Red substrate stock solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13827
Add 10 µL of 10 mM aspartate standard into 990 µL of 1X PBS buffer to get 100 µM aspartate solution (ASP7). Then perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted aspartate standards (ASP6 - ASP1).
PREPARATION OF WORKING SOLUTION
1. Conversion Mix solution (100X):
Add 50 µL of ddH2O into Conversion Mix (Component D) to make 100X Conversion Mix stock solution.
2. Amplite™ Red working solution:
Add 5 mL Assay Buffer (Component C) into one Enzyme Mix 1 bottle (Component B1); mix well. Add 100 μL of ddH2O into one Enzyme Mix 2 vial (Component B2); mix well. Transfer the entire vial (100 μL) of Enzyme Mix 2, 25 uL of 200X Amplite™ Red substrate stock solution, and 50 μL of 100X Conversion Mix solution into the Enzyme Mix 1 bottle and mix well. Note: The working solution is not stable, use it promptly, and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of aspartate standards and test samples in a solid black 96-well microplate. ASP = aspartate standard (ASP1 - ASP7, 0.1 to 100 µM); BL = blank control; TS = test sample.
| BL | BL | TS | TS |
| ASP1 | ASP1 | ... | ... |
| ASP2 | ASP2 | ... | ... |
| ASP3 | ASP3 | ||
| ASP4 | ASP4 | ||
| ASP5 | ASP5 | ||
| ASP6 | ASP6 | ||
| ASP7 | ASP7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| ASP1 - ASP7 | 50 µL | Serial Dilution (0.1 to 100 µM) |
| BL | 50 µL | 1X PBS Buffer |
| TS | 50 | Test Sample |
- Prepare aspartate standards (ASP), blank controls (BL), and test samples (TS) according to 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.
- Add 50 µL of Amplite Red™ working solution into each well of aspartate standard, blank control, and test samples to make the total aspartate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well. Note: Run the aspartate assay at pH 6.5 to 7.0.
- Incubate the reaction mixture at 37°C for 20 - 30 minutes.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em=540/590 nm (cut off=570 nm).
Citations
Authors: Li, Xingwang and Sang, Ziwei and Zhao, Xuejin and Wen, Ying
Journal: Microbial Biotechnology (2024): e70038
Authors: Alexander, Ashley M and Luu, Justin M and Raghuram, Vishnu and Bottacin, Giulia and van Vliet, Simon and Read, Timothy D and Goldberg, Joanna B
Journal: Microbiology (2024): 001445
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