Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Green Fluorescence*
Ordering information
| Price | |
| Catalog Number | |
| Unit Size | |
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Additional ordering information
| Telephone | 1-800-990-8053 |
| Fax | 1-800-609-2943 |
| sales@aatbio.com | |
| International | See distributors |
| Bulk request | Inquire |
| Custom size | Inquire |
| Shipping | Standard overnight for United States, inquire for international |
Spectral properties
| Excitation (nm) | 510 |
| Emission (nm) | 525 |
Storage, safety and handling
| H-phrase | H303, H313, H333 |
| Hazard symbol | XN |
| Intended use | Research Use Only (RUO) |
| R-phrase | R20, R21, R22 |
| UNSPSC | 12171501 |
Alternative formats
| Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence* |
| Overview |  SDSProtocol |
Excitation (nm) 510 | Emission (nm) 525 |
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.
Platform
Fluorescence microplate reader
| Excitation | 490 nm |
| Emission | 525 nm |
| Cutoff | 515 nm |
| Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol Summary
- Prepare AChE standards and/or AChE test samples (50 µL)
- Add AChE working solution (50 µL)
- Incubate at room temperature for 10 - 30 minutes
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
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
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.
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.
Table 2. Reagent composition for each well.
| BL | BL | TS | TS |
| AS1 | AS1 | ... | ... |
| AS2 | AS2 | ... | ... |
| AS3 | AS3 | ||
| AS4 | AS4 | ||
| AS5 | AS5 | ||
| AS6 | AS6 | ||
| AS7 | AS7 |
| Well | Volume | Reagent |
| AS1 - AS7 | 50 µL | Serial Dilution (0.01 to 100 mU/mL) |
| BL | 50 µL | Assay Buffer (Component B) |
| TS | 50 µL | test sample |
- 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.
- 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.
- Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm.
Product Family
| Name | Excitation (nm) | Emission (nm) |
| Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence* | 571 | 584 |
Images
Figure 1. 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 Distribution of Intravenously Administered Acetylcholinesterase Inhibitor and Acetylcholinesterase Activity in the Adrenal Gland: 11C-Donepezil PET Study in the Normal Rat by Tadashi Watabe et al., PLOS, Sep. 2014.
Figure 2. 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: Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides by Li et.al., Toxicology in Vitro. April 2019.
Citations
View all 37 citations: Citation Explorer
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)
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
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
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)
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)
Linarin improves the dyskinesia recovery in Alzheimer's disease zebrafish by inhibiting the acetylcholinesterase activity
Authors: Pan, Hongye and Zhang, Jinghui and Wang, Yangyang and Cui, Keke and Cao, Yueting and Wang, Longhu and Wu, Yongjiang
Journal: Life Sciences (2019)
Authors: Pan, Hongye and Zhang, Jinghui and Wang, Yangyang and Cui, Keke and Cao, Yueting and Wang, Longhu and Wu, Yongjiang
Journal: Life Sciences (2019)
Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides
Authors: Li, Shuaizhang and Zhao, Jinghua and Huang, Ruili and Santillo, Michael F and Houck, Keith A and Xia, Menghang
Journal: Toxicology in Vitro (2019)
Authors: Li, Shuaizhang and Zhao, Jinghua and Huang, Ruili and Santillo, Michael F and Houck, Keith A and Xia, Menghang
Journal: Toxicology in Vitro (2019)
Supplementation of maternal diets with docosahexaenoic acid and methylating vitamins impacts growth and development of fetuses from malnourished gilts
Authors: Lima, Hope K and Lin, Xi and Jacobi, Sheila K and Man, Caolai and Sommer, Jeffrey and Flowers, William and Blikslager, Anthony and Gonzalez, Liara and Odle, Jack
Journal: Current developments in nutrition (2018): nzx006
Authors: Lima, Hope K and Lin, Xi and Jacobi, Sheila K and Man, Caolai and Sommer, Jeffrey and Flowers, William and Blikslager, Anthony and Gonzalez, Liara and Odle, Jack
Journal: Current developments in nutrition (2018): nzx006
Hepatic vagus nerve regulates Kupffer cell activation via α7 nicotinic acetylcholine receptor in nonalcoholic steatohepatitis
Authors: Nishio, Takahiro and Taura, Kojiro and Iwaisako, Keiko and Koyama, Yukinori and Tanabe, Kazutaka and Yamamoto, Gen and Okuda, Yukihiro and Ikeno, Yoshinobu and Yoshino, Kenji and Kasai, Yosuke and others, undefined
Journal: Journal of Gastroenterology (2017): 1--12
Authors: Nishio, Takahiro and Taura, Kojiro and Iwaisako, Keiko and Koyama, Yukinori and Tanabe, Kazutaka and Yamamoto, Gen and Okuda, Yukihiro and Ikeno, Yoshinobu and Yoshino, Kenji and Kasai, Yosuke and others, undefined
Journal: Journal of Gastroenterology (2017): 1--12
Protective Effect of α-Lipoic Acid against α-Cypermethrin-Induced Changes in Rat Cerebellum
Authors: Elsawy, H and Al-Omair, MA and Sedky, A and Al-Otaibi, L
Journal: Journal of Chemical Neuroanatomy (2017)
Authors: Elsawy, H and Al-Omair, MA and Sedky, A and Al-Otaibi, L
Journal: Journal of Chemical Neuroanatomy (2017)
Spirulina maxima Extract Ameliorates Learning and Memory Impairments via Inhibiting GSK-3β Phosphorylation Induced by Intracerebroventricular Injection of Amyloid-β 1--42 in Mice
Authors: Koh, Eun-Jeong and Kim, Kui-Jin and Song, Ji-Hyeon and Choi, Jia and Lee, Hyeon Yong and Kang, Do-Hyung and Heo, Ho Jin and Lee, Boo-Yong
Journal: International Journal of Molecular Sciences (2017): 2401
Authors: Koh, Eun-Jeong and Kim, Kui-Jin and Song, Ji-Hyeon and Choi, Jia and Lee, Hyeon Yong and Kang, Do-Hyung and Heo, Ho Jin and Lee, Boo-Yong
Journal: International Journal of Molecular Sciences (2017): 2401
Application notes
A Library of Well-Defined and Water-Soluble Poly(alkyl phosphonate)s with Adjustable Hydrolysis
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Ameliorative Effect of Novel Vitamin Formula with Herbal Extracts on Scopolamine-Induced Alzheimer's Disease
An Increase in Plasma Homovanillic Acid with Cocoa Extract Consumption Is Associated with the Alleviation of Depressive Symptoms in Overweight or Obese Adults
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Ameliorative Effect of Novel Vitamin Formula with Herbal Extracts on Scopolamine-Induced Alzheimer's Disease
An Increase in Plasma Homovanillic Acid with Cocoa Extract Consumption Is Associated with the Alleviation of Depressive Symptoms in Overweight or Obese Adults
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity