Amplite® Colorimetric Butyrylcholinesterase Activity Assay Kit

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Storage, safety and handling

Intended use

Research Use Only (RUO)

Overview SDS Protocol

Butyrylcholinesterase (EC 3.1.1.8; BChE), also known as pseudocholinesterase or plasma cholinesterase, is mainly synthesized in liver and present in blood. BChE is a nonspecific cholinesterase enzyme and can hydrolyze many different choline esters, serving as the first line of defense against toxic compounds reaching the bloodstream. It has been identified as a clinical biomarker of organophosphate poisoning. Amplite® Colorimetric Butyrylcholinesterase Activity Assay Kit is based on a synthetic butyrylthiocholine-based substrate, which can be hydrolyzed by BChE and produce thiocholine. Thiocholine can react with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) and generates a yellow chromophore that can be detected at 410 nm. The assay is convenient, sensitive and can detect as low as 6 mU/mL in variety of samples.

Platform

Absorbance microplate reader

Absorbance	410 ± 5 nm
Recommended plate	Clear bottom

Components

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

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

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

BChE standard

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.

Images

Figure 1. Butyrylcholinesterase dose response was measured in a white/clear bottom 96-well plate with Amplite® Colorimetric Butyrylcholinesterase Assay Kit using a SpectraMax microplate reader. As low as 6.0 mU/mL of Butyrylcholinesterase can be detected with 10 minutes incubation (n=3).

Citations

View all 37 citations: Citation Explorer

Identification of Compounds for Butyrylcholinesterase Inhibition
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

Cerebrotendinous xanthomatosis--three patients, three faces
Authors: Schumann, F and Steinhagen-Thiessen, E and Kassner, U
Journal: Atherosclerosis (2021): e182--e183

Thiol-ene click reaction-induced fluorescence enhancement by altering the radiative rate for assaying butyrylcholinesterase activity
Authors: Chen, G., Feng, H., Xi, W., Xu, J., Pan, S., Qian, Z.
Journal: Analyst (2019): 559-566

Microwave-Assisted Organic Synthesis, structure-activity relationship, kinetics and molecular docking studies of non-cytotoxic benzamide derivatives as selective butyrylcholinesterase inhibitors
Authors: Wajid, S., Khatoon, A., Khan, M. A., Zafar, H., Kanwal, S., Atta Ur, Rahman, Choudhary, M. I., Basha, F. Z.
Journal: Bioorg Med Chem (2019): 4030-4040

Anti-Alzheimer's multitarget-directed ligands with serotonin 5-HT6 antagonist, butyrylcholinesterase inhibitory, and antioxidant activity
Authors: Marcinkowska, M., Bucki, A., Panek, D., Siwek, A., Fajkis, N., Bednarski, M., Zygmunt, M., Godyn, J., Del Rio Valdivieso, A., Kotanska, M., Kolaczkowski, M., Wieckowska, A.
Journal: Arch Pharm (Weinheim) (2019): e1900041

A randomized crossover trial assessing the effects of acute exercise on appetite, circulating ghrelin concentrations, and butyrylcholinesterase activity in normal-weight males with variants of the obesity-linked FTO rs9939609 polymorphism
Authors: Dorling, J. L., Clayton, D. J., Jones, J., Carter, W. G., Thackray, A. E., King, J. A., Pucci, A., Batterham, R. L., Stensel, D. J.
Journal: Am J Clin Nutr (2019): ersion="1.0" encoding="UTF-8" ?>11406.enlEndN

Effects of simvastatin and fenofibrate on butyrylcholinesterase activity in the brain, plasma, and liver of normolipidemic and hyperlipidemic rats
Authors: Vuksic, A., Lovric, J., Konjevoda, P., Blazevic, N., Bilusic, M., Bradamante, V.
Journal: Arh Hig Rada Toksikol (2019): 30-35

Phytochemical content, antioxidant activity, and enzyme inhibition effect of Salvia eriophora Boiss. & Kotschy against acetylcholinesterase, alpha-amylase, butyrylcholinesterase, and alpha-glycosidase enzymes
Authors: Bursal, E., Aras, A., Kilic, O., Taslimi, P., Goren, A. C., Gulcin, I.
Journal: J Food Biochem (2019): e12776

Structure-activity study of fluorine or chlorine-substituted cinnamic acid derivatives with tertiary amine side chain in acetylcholinesterase and butyrylcholinesterase inhibition
Authors: Gao, X. H., Tang, J. J., Liu, H. R., Liu, L. B., Liu, Y. Z.
Journal: Drug Dev Res (2019): 438-445

Flavonols and 4-thioflavonols as potential acetylcholinesterase and butyrylcholinesterase inhibitors: Synthesis, structure-activity relationship and molecular docking studies
Authors: Mughal, E. U., Sadiq, A., Ashraf, J., Zafar, M. N., Sumrra, S. H., Tariq, R., Mumtaz, A., Javid, A., Khan, B. A., Ali, A., Javed, C. O.
Journal: Bioorg Chem (2019): 103124