Amplite™ Fluorimetric Acetylcholine Assay Kit *Red Fluorescence*

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1e+51e+41000100101- Dose-responseData legend Generated with Quest Graph™ Acetylcholine (uM) RFU Hover mouse to interact
Acetylcholine dose response was measured in a 96-well solid black plate with Amplite™ Fluorimetric Acetylcholine Assay Kit (Cat. # 11403) using a Gemini fluorescence microplate reader (Molecular devices).
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200 Tests 11403 $195


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
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Overview

PlatformsFluorescence microplate reader
Storage Freeze (<-15 °C)
Minimize light exposure
Category Neurobiology
Neurological Enzymes
Related Diagnostic Molecules
Microplate Readers
Acetylcholine and its metabolites are needed for three main physiological purposes: structural integrity and signaling roles for cell membranes, cholinergic neurotransmission (acetylcholine synthesis), and as a major source for methyl groups via its metabolite, trimethylglycine (betaine) that participates in the S-adenosylmethionine synthesis pathways. It plays an important role in the central nervous system as a precursor for acetylcholine and membrane phosphatidylcholine. This Amplite™ Fluorimetric Acetylcholine Assay Kit provides one of the most sensitive methods for the quantifying acetylcholine. The kit uses Amplite Red™ to quantify acetylcholine through choline oxidase-mediated enzyme coupling reactions. The fluorescence intensity of Amplite Red™ is proportional to acetylcholine.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare ACh working solution (50 µL)
  2. Add ACh standards or ACh 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.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
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. Amplite™ Red stock solution (250X):
Add 40 µL of DMSO (Component E) into the vial of Amplite Red™ (Component A) to make 250X Amplite™ Red stock solution.

2. Acetylcholine standard solution (50 mM):
Add 200 µL of ddH2O into the vial of Acetylcholine Standard (Component C) to make 50 mM Acetylcholine standard solution.

Preparation of standard solution
Acetylcholine standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11403

Add 20 µL of 50 mM Acetylcholine standard solution to 980 µL Assay Buffer (Component D) to generate 1000 µM Acetylcholine standard solution. Take 1000 µM Acetylcholine standard and perform 1:10 in Assay Buffer (Component D) to get 100 µM Acetylcholine standard (AS7). Take 100 µM Acetylcholine standard (AS7) and 1:3 serial dilutions to get serially dilited of acetylcholine standard (AS6 - AS1) with Assay Buffer (Component D).  Note: Diluted Acetylcholine standard solution is unstable, and should be used within 4 hours.

Preparation of working solution

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

2. Add 20 μL of 250X Amplite Red™ stock solution into the bottle of Acetylcholine Probe solution to make Acetylcholine (ACh) working solution. Note: This Acetylcholine (ACh) 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 Acetylcholine standards and test samples in a solid black 96-well microplate.  AS= Acetylcholine Standards (AS1 - AS7, 0.14 to 100 µM); 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 (0.14 to 100 µM)
BL 50 µL Assay Buffer
TS 50 µL test sample
  1. Prepare Acetylcholine 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 Acetylcholine (ACh) working solution to each well of Acetylcholine standard, blank control, and test samples to make the total Acetylcholine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Acetylcholine (ACh) 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).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Acetylcholine samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

Figure 1. Acetylcholine dose response was measured in a 96-well solid black plate with Amplite™ Fluorimetric Acetylcholine Assay Kit (Cat. # 11403) using a Gemini fluorescence microplate reader (Molecular devices).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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Resources
 
Safety Data Sheet (SDS)


Documents
1. AssayWise Letters 2012, Vol 1(2)

Certificate of Analysis