Amplite® Colorimetric Acetylcholinesterase Assay Kit

Protocol summary

1. Prepare AChE working solution (50 µL)
2. Add AChE standards or AChE test samples (50 µL)
3. Incubate at room temperature for 10 - 30 minutes
4. Monitor absorbance at 410 ± 5 nm

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

1. DTNB stock solution (20X):
Add 0.6 mL of Assay Buffer (Component B) into the vial of DTNB (Component A) to make 20X DTNB stock solution. Keep from light. Note: DNTB is not easy to dissolve, it is normal to see the cloudiness of the solution. One can use either the supernatant or the mixture for the experiment.

2. Acetylthiocholine stock solution (20X):
Add 0.6 mL of ddH₂O into the vial of Acetylthiocholine (Component C) to make 20X Acetylthiocholine stock solution. 

3. Acetylcholinesterase standard solution (50 U/mL):
Add 100 µL of ddH₂O with 0.1% BSA into the vial of Acetylcholinesterase Standard (Component D) to make 50 U/mL Acetylcholinesterase standard solution.

Add 250 μL of 20X DTNB stock solution and 250 μL of 20X Acetylthiocholine stock solution into 4.5 mL of Assay Buffer (Component B) to make a total volume of 5 mL AChE working solution. Keep from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Table 1. Layout of Acetylcholinesterase standards and test samples in a white/clear bottom 96-well microplate. AS=Acetylcholinesterase Standards (AS1 - AS7, 1 to 1000 mU/mL); BL=Blank Control; TS=Test Samples.

Table 2. Reagent composition for each well.

1. Prepare Acetylcholinesterase 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. Note: Treat cells or tissue samples as desired.
   
   
2. Add 50 µL of AChE working solution to each well of Acetylcholinesterase standard, blank control, and test samples to make the total Acetylcholinesterase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of AChE working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction for 10 to 30 minutes at room temperature, protected from light.
   
   
4. Monitor the absorbance increase with an absorbance microplate reader at 410 ± 5 nm.