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Amplite® Fluorimetric Butyrylcholinesterase Activity Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare BChE standards, dye working solution and test samples
- Add BChE standards or test samples (100 uL)
- Add BChE dye working solution (100 uL)
- Incubate at room temperature for 10-30 minutes
- Monitor fluorescence intensity at Ex/Em=490/525 nm
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
- Thiolite™ Green stock solution (200X):
Add 50 uL of DMSO (Component E) into the vial of Thiolite™ Green (Component A) to make 200X Thiolite™ Green stock solution.
- Butyrylthiocholine (BTC) stock solution (100X):
Add 120 uL of ddH2O into the vial of BTC (Component C) to make 100X BTC stock solution.
- Butyrylcholinesterase (BChE) Standard solution (5 U/mL):
Add 50 uL of ddH2O with 0.1% BSA into the vial of BChE Standard (Component D) to make 5 U/mL BChE standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11407
Add 20 uL of 5 U/mL BChE standard solution to 980 uL of Assay Buffer (Component B) to generate 100 mU/mL BChE standard solution (BS1). Then take 100 mU/mL BChE standard solution (BS1) and perform 1:3 serial dilutions in Assay Buffer(Component B) to get serially diluted BChE standards (BS2 - BS7). Note: Diluted BChE standard solution is unstable and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
BChE dye working solution:
Add 50 uL of 200X Thiolite™ Green stock solution and 100 uL of 100X BTC stock solution into 10 mL of Assay Buffer (Component B) to make a total volume of 10.1 mL BChE dye working solution. Note: Keep it from light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of BChE standards and test samples in a clear bottom 96-well microplate. BS=BChE standards (BS1-BS7, 100 to 0.13 mU/mL); BL=Blank Control; TS=Test Samples
|
BL |
BL |
TS |
TS |
|
BS1 |
BS1 |
… |
… |
|
BS2 |
BS2 |
… |
… |
|
BS3 |
BS3 |
|
|
|
BS4 |
BS4 |
|
|
|
BS5 |
BS5 |
|
|
|
BS6 |
BS6 |
|
|
|
BS7 |
BS7 |
|
|
Table 2. Reagent composition for each well.
|
Well |
Volume |
Reagent |
|
BS1-BS7 |
100 uL |
Serial Dilutions (100 to 0.13 mU/mL) |
|
BL |
100 uL |
Assay Buffer (Component B) |
|
TS |
100 uL |
Test Sample |
- Prepare BChE standards (BS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 uL of reagent per well instead of 100 uL. Note: Treat cells or tissue samples as desired.
- Add 100 uL of BChE dye working solution to each well of BChE standard, blank control, and test samples to make the total assay volume 200 uL/well. For a 384-well plate, add 25 uL of BChE working solution into each well instead, for a total volume of 50 uL/well.
- Incubate the reaction for 10 to 30 minutes at room temperature, protected from light.
- Monitor the fluorescence increase with an fluorescence microplate reader at Ex/Em=490/525 nm.
Citations
Authors: Chen, G., Feng, H., Xi, W., Xu, J., Pan, S., Qian, Z.
Journal: Analyst (2019): 559-566
Authors: Orhan, I. E., Senol Deniz, F. S., Traedal-Henden, S., Ceron-Carrasco, J. P., den Haan, H., Pena-Garcia, J., Perez-Sanchez, H., Emerce, E., Skalicka-Wozniak, K.
Journal: Chem Biodivers (2019): e1900017
Authors: Jasiecki, J., Zuk, M., Krawczynska, N., Jonca, J., Szczoczarz, A., Lew and owski, K., Waleron, K., Wasag, B.
Journal: Chem Biol Interact (2019): 154-157
Authors: Jiang, C. S., Ge, Y. X., Cheng, Z. Q., Wang, Y. Y., Tao, H. R., Zhu, K., Zhang, H.
Journal: Molecules (2019): se name="11406.enl" path="C:\Users\aatbi\Drop
Authors: Maximova, N., Caddeo, G., Zanon, D., Maestro, A., Simeone, R.
Journal: J Clin Med (2019): se name="11406.enl" path="C:\Users\aatbi\Drop
Safety data sheet