Amplite® Fluorimetric Alanine Aminotransferase Assay Kit

Protocol summary

1. Prepare ALT working solution (50 µL)
2. Add ALT standards or test samples (50 µL)
3. Incubate at 37°C for 30 min to 60 min
4. Monitor fluorescence increase at Ex/Em = 540/590 nm

Important notes
Thaw one bottle Component A and B at room temperature before starting the experiment.

1. ALT standard solution:
Add 100 µL DPBS into the vial of ALT Positive Control (Component D) to make 100 U/mL ALT stock solution.

1. NAD solution (100X):
Add 100 µL of ddH2O into the vial of NAD (Component C).

2. ALT Enzyme Mixture solution:
Add 10 mL of ALT Assay Buffer (Component B) into the bottle of ALT Enzyme Mixture (Component A), and mix well.

3. ALT working solution:
Add the whole vial of 100X NAD solution (from 1) into the ALT Enzyme Mixture solution (from 2) to have ALT working solution.

Note: This ALT working solution is enough for two 96-well plates. It is unstable at room temperature, and should be used promptly within 2 hours and avoid exposure to light. Alternatively, one can make a 50X of ALT Enzyme Mixture stock solution by adding 200 μL of H₂O into the bottle of Component A, and then prepare the ALT working solution by mixing the stock solution with assay buffer (Component B) and 100x NAD solution proportionally. 

Table 1. Layout of ALT standards and test samples in a solid black 96-well microplate. ALT = ALT standard (ALT1 - ALT7, 1 to 1000 mU/mL); BL = blank control; TS = test sample.

Table 2. Layout of ALT standards and test samples in a solid black 96-well microplate.

1. Prepare ALT standards (ALT), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according the the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Dilute the test samples to the appropriate concentration range (indicated by the ALT standards) in DPBS buffer with 0.1% BSA if needed.
   
   
2. Add 50 µL of ALT working solution into each well of ALT standard, blank control, and test samples to make the total ALT assay volume of 100 µL/well. For a 384-well microplate, add 25 µL of ALT working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at 37°C for 30 min to 60 minutes, protected from light. Note: The background of Blank Control will increase with time.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off at 570 nm).