Amplite® Fluorimetric L-Aspartate (Aspartic Acid) Assay Kit

Protocol summary

1. Prepare test samples along with diluted aspartate standards (50 µL)
2. Add equal volume of working solution (50 µL)
3. Incubate at 37°C for 20 - 30 minutes
4. 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.

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.

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.

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.

Table 2. Reagent composition for each well.

1. 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.
   
   
2. 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.
   
   
3. Incubate the reaction mixture at 37°C for 20 - 30 minutes.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em=540/590 nm (cut off=570 nm).