Amplite® Colorimetric Asparaginase Activity Assay Kit

1. Prepare the test samples and the serially diluted Aspartate standards (50 μL).

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

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

4. Measure the absorbance at 575 nm.

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

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

   Note: The solution can be stored at -20°C. Avoid repeated freeze/thaw cycles.

1. Reconstitute the Asparaginase Positive Control (Component F) with 100 µL of ddH2O. Mix well by pipetting and store at -20 °C.

   Note: Must be used within 2 months of reconstitution. Avoid freeze/thaw cycles.

1. To prepare a 10 mM Aspartate Standard stock solution, add 100 µL of ddH2O to the vial containing the Aspartate Standard (Component E), and mix well.

   Note: The solution can be aliquoted and stored at -20°C. Avoid freeze/thaw cycles.

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

   Note: The solution can be aliquoted and stored at -20°C. Avoid freeze/thaw cycles.

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

   Note: The solution can be aliquoted and stored at -20°C. Avoid freeze/thaw cycles.

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

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

3. Transfer 50 µL of the Conversion Mix stock solution and 50 µL of the 100X Amplite® Red Substrate stock solution to the same bottle, and mix well.

   Note: This Asparaginase working solution should be freshly prepared before each experiment and protected from light. Due to its instability, it should be used immediately after preparation.

   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. Prepare one or more Asparaginase positive control samples along with the test sample. The recommended dilution factor is 100-1000 fold in 1X PBS. For example, for a 250-fold dilution, mix 4 µL of the Asparaginase stock solution with 996 µL of PBS.

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

Table 2. Reagent composition for each well.

1. Prepare Aspartate standards (STD1-7), blank controls (BL), Asparaginase Positive Control, and test samples (TS) as outlined 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 Asparaginase Working Solution to each well containing the Aspartate standard, blank control, Asparaginase Positive Control, and test samples. For a 384-well plate, add 25 µL of the Asparaginase Working Solution to each well instead.

3. Incubate at room temperature for 10-30 minutes, protected from light.

4. Monitor the absorbance intensity with an absorbance microplate reader at 575 nm.