Amplite® Fluorimetric L-Alanine Assay Kit

Protocol summary

1. Prepare test samples along with serially diluted L-Alanine standards (50 µL)
2. Add equal volume of L-Alanine working solution (50 µL)
3. Incubate at 37°C for 30 minutes to 1 hour
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
To achieve the best result, it’s strongly recommended to use the black plates. Thaw kit components at room temperature before use.

1. Quest Fluor™ L-Alanine Sensor stock solution (200X):
Add 55 µL of DMSO (Component E) into Quest Fluor™ L-Alanine Sensor (Component A) to make 200X Quest Fluor™ L-Alanine Sensor stock solution.

2. L-Alanine standard solution (1 mM):
Add 10 µL of 100 mM L-alanine (Component D) into 990 µL PBS (pH 7.0) to get 1 mM L-alanine solution.

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

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

3. Transfer entire vial (100 μL) of Enzyme Mix 2 and 25 uL of 200X L-alanine sensor stock solution into the Enzyme Mix 1 bottle; mix well. Note: The working solution is not stable. Use promptly and avoid direct exposure to light.

Table 1. Layout of L-alanine standards and test samples in a solid black 96-well microplate. AS = L-Alanine standard (AS1 - AS7, 1.5 to 100 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 mixture at 37°C for 30 minutes to 1 hour.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (cut off: 570 nm).