Amplite® Fluorimetric Asparaginase Activity Assay Kit

Thaw all the kit components at room temperature before starting the experiment. 

1. Prepare test samples and serially dilute Aspartate or Asparaginase standards (50 μL).

2. Add the Asparaginase working solution (50 μL).

3. Incubate at 37 °C for 10-30 minutes.

4. Monitor the fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm).

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

1. To prepare a 100X Amplite® Red Substrate stock solution, add 60 µL of DMSO (Component G) to the vial of Amplite® Red Substrate (Component A).

1. To prepare a 50X Enzyme Mix 2 stock solution, add 100 µL of ddH2O to the Enzyme Mix 2 vial (Component B2).

1. To prepare a 100X Conversion Mix stock solution, add 50 µL of ddH2O to the Conversion Mix vial (Component D).

1. To prepare at 10 mM aspartate standard solution, add 100 µL of ddH2O to the Aspartate Standard vial (Component E).

1. Reconstitute Asparaginase Positive Control (Component F) with 100 µL of ddH2O, and mix well. Aliquot the solution and store it at -20 °C.

1. Add 5 mL of Assay Buffer (Component C) to the Enzyme Mix 1 bottle (Component B1) and mix well.

2. Add 100 μL of Enzyme Mix 2 stock solution to the same bottle and mix well.

3. Add 50 μL of the 100X Conversion Mix stock solution to the same bottle and mix well.

4. Add 50 µL of the Amplite® Red Substrate stock solution to the same bottle and mix well.

   Note: The working solution is not stable, use it promptly, and avoid direct exposure to light.

   Note: Alternatively, one can make a 50X Enzyme Mix 1 stock solution by adding 100 μL of ddH2O into the bottle of Enzyme Mix 1 (Component B1) and then prepare the Asparaginase working solution by mixing the Enzyme Mix 1 stock solution with other components listed above in the 'Asparaginase Working Solution' proportionally.

1. To prepare one or more Asparaginase positive controls alongside the test sample, dilute the Asparaginase Positive Control stock solution using a dilution factor between 100 and 1000 times in 1X PBS. For example, for a 250-fold dilution, mix 4 µL of Asparaginase Positive Control stock solution with 996 µL of 1X PBS.

Table 1. Layout of Aspartate standards and test samples in a 96-well clear bottom white microplate. (STD = Aspartate Standards (STD1-STD7, 6.25 to 400 µM), BL = Blank Control, TS = Test Samples)

Table 2. Reagent composition for each well.

1. Prepare the Aspartate standards (STD 1-STD 7), blank controls (BL), Asparaginase Positive Control, and test samples (TS) according to the layout provided in Tables 1 and 2. When using a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

2. Add 50 µL of Asparaginase Working Solution to each well of the blank control, Asparaginase Positive Control, and test samples. If using a 384-well plate, add 25 µL of Asparaginase Working Solution to each well instead.

3. Incubate at 37 °C for 10-30 minutes, protected from light.

4. Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 540 nm/ 590 nm (Cutoff = 570 nm).