Amplite® Colorimetric L-Alanine Assay Kit

1. Prepare L-Alanine standards or test samples (50 µL)
2. Add L-Alanine working solution (50 µL)

3. Incubate at 37°C for 1 hour to 2 hours

4. Monitor absorbance intensity at 575 nm

To achieve the best result, it is strongly recommended to use the white clear plates. Thaw kit components at room temperature before use.

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 50 µL of DMSO (Component E) into Quest Fluor™ L-Alanine Sensor (Component A) to make 200X Quest Fluor™ L-Alanine Sensor stock solution.

Add 10 µL of 100 mM L-Alanine Standard (Component D) into 490 µL of PBS (pH 7.0) to get 2 mM L-Alanine standard solution. Add 7.5 µL of 100 mM L-Alanine Standard (Component D) into 492.5 µL of PBS (pH 7.0) to get 1.5 mM L-Alanine standard solution.

Use 2 mM L-Alanine standard stock solution and perform 1:2 serial dilutions in PBS to get serially diluted L-Alanine standards (2000, 1000, 500, 250, 125 uM). Use 1.5 mM L-Alanine standard stock solution and perform 1:2 serial dilutions in PBS to get serially diluted L-Alanine standards (1500, 750 uM).

1. Add 5 mL Assay Buffer (Component C) into one Enzyme Mix1 bottle (Component B1) and mix well.

2. Add 100 μL of ddH₂O into one Enzyme Mix2 vial (Component B2) and mix well.

3. Transfer entire vial (100 μL) of Enzyme Mix2 and 25 uL of 200X L-Alanine Sensor stock solution into the Enzyme Mix1 bottle and mix well to make L-Alanine working solution. Note: L-Alanine working solution is not stable - use it promptly and avoid direct exposure to light.

Table 1. Layout of L-Alanine standards and test samples in a white clear 96-well microplate. AS= L-Alanine Standard (AS1 - AS7, 125 to 2000 µM), BL=Blank Control, TS=Test Sample.

Table 2. Reagent composition for each well.

1. Prepare L-Alanine standards (AS), blank controls (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 L-Alanine working solution to each well of L-Alanine standard, blank control, and test samples to make the total L-Alanine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-Alanine working solution into each well instead, for a total volume of 50 µL/well. Note: Run the L-Alanine assay at pH 6.5 to 7.0.

3. Incubate the reaction at 37°C for 1 hour to 2 hours.

4. Monitor the absorbance increase with an absorbance plate reader at 575 nm.