Amplite® Colorimetric Acetylcholinesterase Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | ![]() ![]() |
Platform
Absorbance microplate reader
Absorbance | 410 ± 5 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare AChE working solution (50 µL)
- Add AChE standards or AChE test samples (50 µL)
- 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
1. DTNB stock solution (20X):
Add 0.6 mL of Assay Buffer (Component B) into the vial of DTNB (Component A) to make 20X 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.
2. Acetylthiocholine stock solution (20X):
Add 0.6 mL of ddH2O into the vial of Acetylthiocholine (Component C) to make 20X Acetylthiocholine stock solution.
3. Acetylcholinesterase standard solution (50 U/mL):
Add 100 µL of ddH2O with 0.1% BSA into the vial of Acetylcholinesterase Standard (Component D) to make 50 U/mL Acetylcholinesterase standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11400
Add 20 µL of 50 U/mL Acetylcholinesterase standard solution to 980 µL of Assay Buffer (Component B) to generate 1000 mU/mL Acetylcholinesterase standard solution (AS7). Then take 1000 mU/mL Acetylcholinesterase standard solution (AS7) and perform 1:3 serial dilutions in Assay Buffer (Component B) to get 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 250 μL of 20X DTNB stock solution and 250 μL of 20X Acetylthiocholine stock solution into 4.5 mL of Assay Buffer (Component B) to make a total volume of 5 mL AChE working solution. Keep from light.
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 white/clear bottom 96-well microplate. AS=Acetylcholinesterase Standards (AS1 - AS7, 1 to 1000 mU/mL); BL=Blank Control; TS=Test Samples.
BL | BL | TS | TS |
AS1 | AS1 | ... | ... |
AS2 | AS2 | ... | ... |
AS3 | AS3 | ||
AS4 | AS4 | ||
AS5 | AS5 | ||
AS6 | AS6 | ||
AS7 | AS7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
AS1 - AS7 | 50 µL | Serial Dilutions (1 to 1000 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. Note: Treat cells or tissue samples as desired.
- 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 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
![Acetylcholinesterase dose response was measured in a white/clear bottom 96-well plate with Amplite® Colorimetric Acetylcholinesterase Assay Kit using a SpectraMax microplate reader (Molecular devices). As low as 0.1 mU/well of Acetylcholinesterase can be detected with 30 minutes incubation(n=3).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-colorimetric-acetylcholinesterase-assay-kit%2Fgraph-for-amplite-colorimetric-acetylcholinesterase-assay-kit_eucbu.webp&w=3840&q=75)
![Ca<sup>2+</sup> response of reticulocytes to LPA stimulation.(A) A representative image of a new methylene blue staining of RBCs from a BALB/c mouse after induction of reticulocytosis. The coloured regions depict reticulocytes analysed in (B) and (C). The arrowheads point to lysed RBCs. (B) Image of Fluo-4 loaded live RBCs. The coloured regions are transferred from (A). The dashed grey circle labels a responding RBC. (C) Intensity traces for Ca<sup>2+</sup> content of the cells marked in (B) stimulated with LPA. (D) Statistics of the maximal response of reticulocytes and the entire RBCs population without reticulocytes (referred to as erythrocytes) under control conditions and for 5 µM LPA stimulation. The numbers below the boxes give the cell numbers taken from three mice. (E) AChE activity in reticulocytes and erythrocytes with and without stimulation with 5 µM LPA for 15 min. The measurements comprise of a colorimetric assay based on 2×106 cells per measurement and the data is the average of 5 mice. *Acetylcholinesterase (AChE) activity of 2×106 RBCs of each population were performed using a colorimetric AChE assay kit (Amplite, AAT Bioquest, USA) following the manufacturers instructions. Source: Graph from <strong>Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation</strong> by Jue Wang et al., <em>PLOS</em>, Jun. 2013.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-colorimetric-acetylcholinesterase-assay-kit%2Ffigure-for-amplite-colorimetric-acetylcholinesterase-assay-kit_U2agH.jpg&w=3840&q=75)
![Hippocampal levels of the AChE, CREB and neurotrophic proteins. AChE activity (A) in the hippocampus was measured with an ELISA kit. The western blotting assay for the phosphorylated CREB, BDNF, and NGF levels in hippocampal tissues (B) and BDNF levels in HT22 cell (C), and their relative intensities (D) were shown. The data are expressed as the means ± SD (n = 7 or 3). #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with the vehicle group; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group. *The acetylcholinesterase (AChE) activity in the hippocampus was determined using an AChE activity assay kit (AAT Bioquest; Sunnyvale, CA, USA) according to the manufacturer’s protocol. The absorbance at 410 nm was measured using a UV spectrophotometer. Source: Graph from <strong>Gongjin-Dan Enhances Hippocampal Memory in a Mouse Model of Scopolamine-Induced Amnesia</strong> by Jin-Seok Lee et al., <em>PLOS</em>, Aug. 2016. ](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-colorimetric-acetylcholinesterase-assay-kit%2Ffigure-for-amplite-colorimetric-acetylcholinesterase-assay-kit_2x5wZ.jpg&w=3840&q=75)
![AChE activity, mAChR1, phosphorylated CREB, and BDNF protein and gene expression as possible mechanisms of PNE action. (A) AChE activity in the hippocampus (n = 7). (B) Phosphorylated CREB and BDNF levels in the hippocampus determined by Western blotting (n = 7). (C) Quantification of phosphorylated CREB/CREB intensity. (D) Quantification of BDNF/β-actin intensity. (E) Alterations in the expression of CREB1, mAChR1, BDNF, CBP and iNOS determined by real time-PCR (n = 6). Gene expression was normalized to that of β-actin. Data are expressed as means ± SD. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the naïve group; *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group. PNE; pine needle extract, THA; tacrine. Source: <strong>Hippocampal memory enhancing activity of pine needle extract against scopolamine-induced amnesia in a mouse model </strong>by Lee et al., <em>Scientific Reports</em>, May 2015.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-colorimetric-acetylcholinesterase-assay-kit%2Ffigure-for-amplite-colorimetric-acetylcholinesterase-assay-kit_kfr5e.jpg&w=3840&q=75)
![The 70% ethanol extract of Spirulina maxima (SM70EE) ameliorates learning and memory impairments by inhibiting the amyloid-β (Aβ) accumulation induced by intracerebroventricular injection of Aβ1–42 in mice. Mouse hippocampal lysates were subjected to AChE assay to investigate AChE activity (n = 4 per group). Results were analyzed by one-way analysis of variance and Duncan’s multiple range test. Source: <b>Spirulina maxima Extract Ameliorates Learning and Memory Impairments via Inhibiting GSK-3β Phosphorylation Induced by Intracerebroventricular Injection of Amyloid-β 1–42 in Mice</b> by Koh et.al., <em>Int. J. Mol. Sci.</em> Nov. 2017.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-colorimetric-acetylcholinesterase-assay-kit%2Ffigure-for-amplite-colorimetric-acetylcholinesterase-assay-kit_24vMj.png&w=3840&q=75)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-colorimetric-acetylcholinesterase-assay-kit%2Ffigure-for-amplite-colorimetric-acetylcholinesterase-assay-kit_OfE6S.png&w=3840&q=75)
Citations
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: Desprez, Charlotte and Danovi, Davide and Knowles, Charles H and Day, Richard M
Journal: Journal of Tissue Engineering (2023): 20417314221139794
Authors: Jamadagni, Shrirang B and Ghadge, Pooja M and Tambe, Mukul S and Srinivasan, Marimuthu and Prasad, Goli Penchala and Jamadagni, Pallavi S and Prasad, Shyam Baboo and Pawar, Sharad D and Gurav, Arun M and Gaidhani, Sudesh N and others,
Journal: Pharmacognosy Magazine (2023): 09731296221145063
Authors: Shaban, Nadia Z and Abd El-Kader, Sara E and Mogahed, Fayed AK and El-Kersh, Mohamed AL and Habashy, Noha H
Journal: Scientific Reports (2021): 1--18
Authors: Tsay, Gregory J and Lin, Yu-Ta and Hsu, Chia-Hong and Tang, Feng-Yao and Kuo, Yueh-Hsiung and Chao, Che-Yi
Journal: Biochemistry and Biophysics Reports (2021): 101020
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: Chandran, Remya and Vijayan, Dileep and Reddy, Eeda Koti and Kumar, Mantosh and Kesavan, Lakshmi and Jacob, Reena and Ayyiliyath, Sajith and Variyar, Jayadevi and Anwar, Shaik and Zhang, Kam and others,
Journal: bioRxiv (2021)
Authors: Lim, Gyeong Taek and You, Dong Gil and Han, Hwa Seung and Lee, Hansang and Shin, Sol and Oh, Byeong Hoon and Kumar, EK Pramod and Um, Wooram and Kim, Chan Ho and Han, Seungsu and others,
Journal: Journal of extracellular vesicles (2021): e12077
Authors: Li, Shuaizhang and Zhao, Jinghua and Huang, Ruili and Travers, Jameson and Klumpp-Thomas, Carleen and Yu, Wenbo and MacKerell Jr, Alexander D and Sakamuru, Srilatha and Ooka, Masato and Xue, Fengtian and others,
Journal: Environmental health perspectives (2021): 047008
Authors: Yuan, Q and Lin, ZX and Wu, W and Albert, WN and Zee, BCY
Journal: Hong Kong Med J (2020)
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Application notes
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