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Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Green Fluorescence*

Acetylcholinesterase, also known as AChE, is an enzyme that degrades (through its hydrolytic activity) the neurotransmitter acetylcholine, producing choline and an acetate group. It is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system, where its activity serves to terminate synaptic transmission. AChE has a very high catalytic activity- each molecule of AChE degrades about 5000 molecules of acetylcholine per second. Acetylcholinesterase is also found on the red blood cell membranes, where it constitutes the Yt blood group antigen. Acetylcholinesterase exists in multiple molecular forms, which possess similar catalytic properties, but differ in their oligomeric assembly and mode of attachment to the cell surface. This Amplite® Fluorimetric Acetylcholinesterase Assay Kit provides the most sensitive method for the detecting AChE activity. The kit uses our outstanding Thiolite Green™ to quantify the thiocholine produced from the hydrolysis of acetylthiocholine by AChE. The fluorescence intensity of Thiolite Green™ is proportional to the formation of thiolcholine, thus the AChE activity.

Example protocol

AT A GLANCE

Protocol Summary
  1. Prepare AChE standards and/or AChE test samples (50 µL)
  2. Add AChE working solution (50 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 490/525 nm 
Important      Thaw all the kit components at room temperature before starting the experiment

CELL PREPARATION

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

PREPARATION OF STOCK SOLUTIONS

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.

1. Thiolite™ Green stock solution (200X)
Add 50 µL of DMSO (Component E) into the vial of Thiolite™ Green (Component A) to make 200X Thiolite™ Green stock solution.

2. Acetylthiocholine stock solution (500X)
Add 0.6 mL of ddH2O into the vial of Acetylthiocholine (Component C).

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

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11401

Acetylcholinesterase standard
Add 20 µL of 50 U/mL Acetylcholinesterase standard solution to 980 µL Assay Buffer (Component C) to generate 1000 mU/mL Acetylcholinesterase standard solution. Take 1000 mU/mL Acetylcholinesterase standard solution to perform 1:10 to get 100 mU/mL Acetylcholinesterase standard solution(AS7). Then perform 1:3 serial dilution to obtain remaining serially diluted acetylcholinesterase standards (AS6 - AS1). Note: Diluted acetylcholinesterase standard solution is unstable and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 10 μL of Acetylthiocholine stock solution (500X) and 25 μL of Thiolite™ Green stock solution (200X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.03 mL Acetylcholinesterase(AChE) working solution.
Note     The AChE working solution is not stable and needs to be used within 30 minutes. Keep from light. 

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of acetylcholinesterase standards and test samples in a solid black 96-well microplate. AS= Acetylcholinesterase Standards (AS1 - AS7, 0.01 to 100 mU/mL), BL=Blank Control, TS=Test Samples.
BLBLTSTS
AS1AS1......
AS2AS2......
AS3AS3
AS4AS4
AS5AS5
AS6AS6
AS7AS7
Table 2. Reagent composition for each well.
WellVolumeReagent
AS1 - AS750 µLSerial Dilution (0.01 to 100 mU/mL)
BL50 µLAssay Buffer (Component B)
TS50 µLtest sample
  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.
  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 at room temperature for 10 to 30 minutes, protected from light.
  4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm. 

Spectrum

Product family

NameExcitation (nm)Emission (nm)
Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*571584

Citations

View all 38 citations: Citation Explorer
Aluminum chloride and D-galactose induced a zebrafish model of Alzheimer's disease with cognitive deficits and aging
Authors: Luo, Li and Yan, Tao and Yang, Le and Zhao, Minggao
Journal: Computational and Structural Biotechnology Journal (2024)
Identification of Potent and Selective Acetylcholinesterase/Butyrylcholinesterase Inhibitors by Virtual Screening
Authors: Xu, Tuan and Li, Shuaizhang and Li, Andrew J and Zhao, Jinghua and Sakamuru, Srilatha and Huang, Wenwei and Xia, Menghang and Huang, Ruili
Journal: Journal of Chemical Information and Modeling (2023)
Imaging mass spectrometry to visualise increased acetylcholine in lungs of asthma model mice
Authors: Matsuda, Takeshi and Suzuki, Yuzo and Fujisawa, Tomoyuki and Suga, Yasunori and Saito, Nobuyuki and Suda, Takafumi and Yao, Ikuko
Journal: Analytical and Bioanalytical Chemistry (2020): 1--15
Potential Effects of Indole-3-Lactic Acid, a Metabolite of Human Bifidobacteria, on NGF-Induced Neurite Outgrowth in PC12 Cells
Authors: Wong, Chyn Boon and Tanaka, Azusa and Kuhara, Tetsuya and Xiao, Jin-zhong
Journal: Microorganisms (2020): 398
Fenobucarb-induced developmental neurotoxicity and mechanisms in zebrafish
Authors: Zhu, Xiao-Yu and Wu, Yu-Ying and Xia, Bo and Dai, Ming-Zhu and Huang, Yan-Feng and Yang, Hua and Li, Chun-Qi and Li, Ping
Journal: Neurotoxicology (2020)
Page updated on November 20, 2024

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Catalog Number11401
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Request quotation

Spectral properties

Excitation (nm)

505

Emission (nm)

524

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components

The relationships among Vt by Logan plot analysis, SUV at 30 min, radioactivity count by well counter, and AChE activity by fluorometric assay. *The AChE activities and ACh levels were quantified using the AmpliteTM Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Inc). Source: Table from <strong>Distribution of Intravenously Administered Acetylcholinesterase Inhibitor and Acetylcholinesterase Activity in the Adrenal Gland: 11C-Donepezil PET Study in the Normal Rat</strong> by Tadashi Watabe et al., <em>PLOS</em>, Sep. 2014.&nbsp;
The relationships among Vt by Logan plot analysis, SUV at 30 min, radioactivity count by well counter, and AChE activity by fluorometric assay. *The AChE activities and ACh levels were quantified using the AmpliteTM Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Inc). Source: Table from <strong>Distribution of Intravenously Administered Acetylcholinesterase Inhibitor and Acetylcholinesterase Activity in the Adrenal Gland: 11C-Donepezil PET Study in the Normal Rat</strong> by Tadashi Watabe et al., <em>PLOS</em>, Sep. 2014.&nbsp;
The relationships among Vt by Logan plot analysis, SUV at 30 min, radioactivity count by well counter, and AChE activity by fluorometric assay. *The AChE activities and ACh levels were quantified using the AmpliteTM Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Inc). Source: Table from <strong>Distribution of Intravenously Administered Acetylcholinesterase Inhibitor and Acetylcholinesterase Activity in the Adrenal Gland: 11C-Donepezil PET Study in the Normal Rat</strong> by Tadashi Watabe et al., <em>PLOS</em>, Sep. 2014.&nbsp;
Concentration-response curves of human AChE assays. There were concentration-dependent inhibition curves in 1536-well plates treated with two positive controls, chlorpyrifos oxon and BW284C5. Two methods were used to measure AChE activity including colorimetric (A, B) and fluorescent methods (C, D). Each value represents the mean ± SD of three independent experiments. Amplite Colorimetric Acetylcholinesterase Assay kit (Ellman assay) and Amplite Fluorimetric Acetylcholinesterase Assay kit (Green Fluorescence) were purchased from AAT Bioquest, Inc. Source: <b>Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides</b> by Li et.al., <em>Toxicology in Vitro</em>. April 2019.

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
Amplite® Reagents and Assay Kits
Cell Signaling Pathways

Alternative formats

Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*