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Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*

Acetylcholinesterase dose response was measured in a solid black 96-well plate with Amplite® Fluorimetric Acetylcholinesterase Assay Kit using a Gemini fluorescence microplate reader (Molecular devices).
Acetylcholinesterase dose response was measured in a solid black 96-well plate with Amplite® Fluorimetric Acetylcholinesterase Assay Kit using a Gemini fluorescence microplate reader (Molecular devices).
Acetylcholinesterase dose response was measured in a solid black 96-well plate with Amplite® Fluorimetric Acetylcholinesterase Assay Kit using a Gemini fluorescence microplate reader (Molecular devices).
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Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Excitation (nm)
571
Emission (nm)
584
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 one of the most sensitive methods for the detecting AChE activity. The kit uses Amplite® Red to quantify the choline produced from the hydrolysis of acetylcholine by AChE through choline oxidase-mediated enzyme coupling reactions. The fluorescence intensity of Amplite® Red™ is proportional to the formation of choline, thus the AChE activity.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare AChE working solution (50 µL)
  2. Add AChE standards or AChE test samples (50 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em =540/590 nm (Cutoff = 570 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.

1. AmpliteTM Red stock solution (250X):
Add 40 µL of DMSO (Component G) into the vial of AmpliteTM Red (Component A) to make 250X AmpliteTM Red stock solution.  Note: Avoid exposure to light. 

2. Acetylcholinesterase standard solution (50 units/mL):
Add 100 µL of Assay Buffer (Component E) into the vial of Acetylcholinesterase Standard (Component D) to make 50 Units/mL Acetylcholinesterase standard solution. 

3. Acetylcholine stock solution (1000X):
Add 100 µL of Assay Buffer (Component E) into the vial of Acetylcholine (Component C) to make a 1000X Acetylcholine stock solution.

PREPARATION OF STANDARD SOLUTION

Acetylcholinesterase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11402

Add 20 µL of 50 Units/mL Acetylcholinesterase standard solution to 980 µL of Dilution Buffer (Component F) to generate 1000 mU/mL Acetylcholinesterase standard solution. Take 1000 mU/mL Acetylcholinesterase standard and perform 1:10 in Dilution Buffer (Component F) to get 100 mU/mL Acetylcholinesterase standard (AS7). Then take 100 mU/mL Acetylcholinesterase standard (AS7) and perform 1:3 serial dilutions to get serially diluted Acetylcholinesterase standard (AS6 - AS1) with Dilution Buffer (Component F). Note: Diluted Acetylcholinesterase standard solution is unstable and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

1. Add 5 mL of Assay Buffer (Component E) to the bottle of Acetylcholinesterase Probe (Component B) and mix well.

2. Add 5 μL of 1000X Acetylcholine stock solution into the bottle of Acetylcholinesterase Probe mixture and mix well.

3. Add 20 μL of 250X AmpliteTM Red stock into this bottle of Acetylcholinesterase Probe mixture to make Acetylcholinesterase (AChE) working solution.  Note: This Acetylcholinesterase (AChE) working solution should be used promptly and kept from light.  The assay background would increase with longer storage time.

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

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Acetylcholinesterase standards and test samples in a solid black 96-well microplate. AS= Acetylcholinesterase Standards (AS1 - AS7, 0.14 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 Dilutions (0.14 to 100 mU/mL)
BL50 µLDilution Buffer (Component F)
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. Note: Treat cells or tissue samples as desired.

  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 = 540/590 nm (Cutoff = 570 nm).

Spectrum


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spectrum

Spectral properties

Excitation (nm)571
Emission (nm)584

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Citations


View all 32 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)
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
Identification of acetylcholinesterase inhibitors using homogenous cell-based assays in quantitative high-throughput screening platforms
Authors: Li, Shuaizhang and Huang, Ruili and Solomon, Samuel and Liu, Yitong and Zhao, Bin and Santillo, Michael F and Xia, Menghang
Journal: Biotechnology journal (2017): 1600715
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)
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
Dietary and donepezil modulation of mTOR signaling and neuroinflammation in the brain
Authors: Dasuri, Kalavathi and Zhang, Le and Kim, Sun OK Fern and ez , undefined and Bruce-Keller, Annadora J and Keller, Jeffrey N
Journal: Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease (2016): 274--283
Anethole restores delayed gastric emptying and impaired gastric accommodation in rodents
Authors: Asano, Teita and Aida, Shuji and Suemasu, Shintaro and Mizushima, Tohru
Journal: Biochemical and biophysical research communications (2016): 125--130
Dissolved organic matter or salts change the bioavailability processes and toxicity of the nanoscale tetravalent lead corrosion product PbO2 to medaka fish
Authors: Chiang, Chun-Wei and Ng, Ding-Quan and Lin, Yi-Pin and Chen, Pei-Jen
Journal: Environmental Science & Technology (2016): 11292--11301
Gongjin-Dan Enhances Hippocampal Memory in a Mouse Model of Scopolamine-Induced Amnesia
Authors: Lee, Jin-Seok and Hong, Sung-Shin and Kim, Hyeong-Geug and Lee, Hye-Won and Kim, Won-Yong and Lee, Sam-Keun and Son, Chang-Gue
Journal: PloS one (2016): e0159823
Functionalised dihydroazo pyrimidine derivatives from Morita--Baylis--Hillman acetates: synthesis and studies against acetylcholinesterase as its inhibitors
Authors: Reddy, Eeda Koti and Remya, C and Sajith, Ayyiliath M and Dileep, KV and Sadasivan, C and Anwar, Shaik
Journal: RSC Advances (2016): 77431--77439