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

1. Prepare test samples (50 µL) along with serially diluted aspartate standards (50 µL)
2. Add equal volume of working solution (50 µL)

3. Incubate at 37 °C for 30 - 60 minutes

4. Monitor absorbance increase at OD of 575±5 nm

Thaw kit components at room temperature before use. To achieve the best results, it’s recommended to use the clear bottom plates.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 100 µL of ddH₂O into Aspartate Standard vial (Component E) to make 10 mM aspartate standard solution.

Add 50 µL of DMSO (Component F) into Amplite™ Red substrate (Component A) to make 200X Amplite™ Red substrate stock solution.
Note        Store unused 200X Amplite™ Red stock solution at -20 ^oC, avoid light and repeated freeze-thaw cycles.

Add 50 µL of ddH₂O into Conversion Mix (Component D) to make 100X Conversion Mix solution.

Add 5 mL Assay Buffer (Component C) into one Enzyme Mix 1 bottle (Component B1) and mix well. Add 100 μL of ddH₂O into one Enzyme Mix 2 vial (Component B2) and mix well. Transfer entire vial (100 μL) of Enzyme Mix 2, 25 μL 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 clear bottom 96-well microplate. ASP= Aspartate Standard (ASP1 - ASP7, 12.5 to 100 µM), BL=Blank Control (1×PBS buffer), TS=Test Sample.

Table 2. Reagent composition for each well.

1. Prepare aspartate standards (ASP), blank control (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.
2. Add 50 µL of Amplite™ Red working solution to 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 Amplite™ Red 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 30 - 60 minutes.

4. Monitor the absorbance increase with an absorbance plate reader with path check at OD of 575 nm.