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Amplite® Colorimetric Butyrylcholinesterase Activity Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare BChE standards, test samples and dye working solution
- Add BChE standards or test samples (100 uL)
- Add BChE dye working solution (100 uL)
- Incubate at room temperature for 10-30 minutes
- Monitor absorbance at 410 ± 5 nm
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
- DTNB stock solution (10X):
Add 1.2 mL of Assay Buffer (Component B) into the vial of DTNB (Component A) to make 10X 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.
- 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 (20 U/mL):
Add 50 uL of ddH2O with 0.1% BSA into the vial of Butyrylcholinesterase Standard (Component D) to make 20 U/mL butyrylcholinesterase Standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11406
Add 20 uL of 20 U/mL BChE standard solution to 980 uL of Assay Buffer (Component B) to generate 400 mU/mL BChE standard solution (BS1). Then take 400 mU/mL BChE standard solution (BS7) and perform 1:2 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 1.0 mL of 10 X DTNB stock solutions and 100 μL of 100X BTC stock solution into 9 mL of Assay Buffer (Component B) to make a total volume of 10.1 mL BChE dye working solution. Keep away 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, 400 to 6.5 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 (400 to 6.5 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 absorbance increase with an absorbance microplate reader at 410 ± 5 nm.
Citations
Authors: Schumann, F and Steinhagen-Thiessen, E and Kassner, U
Journal: Atherosclerosis (2021): e182--e183
Authors: Li, Shuaizhang and Li, Andrew J and Travers, Jameson and Xu, Tuan and Sakamuru, Srilatha and Klumpp-Thomas, Carleen and Huang, Ruili and Xia, Menghang
Journal: SLAS DISCOVERY: Advancing the Science of Drug Discovery (2021): 24725552211030897
Authors: Gao, X. H., Tang, J. J., Liu, H. R., Liu, L. B., Liu, Y. Z.
Journal: Drug Dev Res (2019): 438-445
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
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