Amplite® Colorimetric Acetylcholinesterase Assay Kit

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).

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.

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.

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.

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Overview SDS Protocol

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® Colorimetric Acetylcholinesterase Assay Kit provides a convenient method for the detecting AChE activity. The kit uses DTNB to quantify the thiolcholine produced from the hydrolysis of acetylthiolcholine by AChE. The absoprtion intensity of DTNB adduct is proportional to the formation of thiolcholine, thus the AChE activity.

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

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. 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

Acetylcholinesterase standard

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

Figure 1. 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).

Figure 2. Ca²⁺ 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 Ca²⁺ 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 Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation by Jue Wang et al., PLOS, Jun. 2013.

Figure 3. 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 Gongjin-Dan Enhances Hippocampal Memory in a Mouse Model of Scopolamine-Induced Amnesia by Jin-Seok Lee et al., PLOS, Aug. 2016.

Figure 4. 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: Hippocampal memory enhancing activity of pine needle extract against scopolamine-induced amnesia in a mouse model by Lee et al., Scientific Reports, May 2015.

Citations

View all 58 citations: Citation Explorer

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Identification of Compounds for Butyrylcholinesterase Inhibition
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Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition
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Journal: Environmental health perspectives (2021): 047008

Long-Term Tolerance Acquisition and Changes in Acetylcholinesterase Activity in Three Cladoceran Species After a 48-H Pulsed Exposure to Pirimicarb
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