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Amplite® Ethanol Quantitation Kit

Ethanol dose response was measured with Amplite® Ethanol Quantitation Kit on a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Ethanol dose response was measured with Amplite® Ethanol Quantitation Kit on a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Ethanol dose response was measured with Amplite® Ethanol Quantitation Kit on a solid black 96-well plate 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


Excitation (nm)
Emission (nm)
The ability to rapidly perform quantitative measurements of ethanol is highly desirable in life science research, clinical evaluations, and food and drug industries. This non-radioactive ethanol assay is based on the oxidation of ethanol by alcohol oxidase. The kit uses our Amplite®Red that makes the kit recordable in a dual mode, either fluorimetric or colorimetric readout. The kit provides all the essential components with an optimized assay protocol. The assay is robust, sensitive, and can be readily adapted for high throughput assays in a wide variety of applications that require the measurement of ethanol. The assay can be completed within 30 minutes.


Absorbance microplate reader

Absorbance576 ± 5 nm
Recommended plateClear bottom

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare Ethanol working solution (50 µL)
  2. Add Ethanol standards or test samples (50 µL)
  3. Incubate at room temperature for 5 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw all the kit components to room temperature before starting the experiment.


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™ Ethanol Reagent stock solution (250X):
Add 40 µL of DMSO (Component D) into the vial of Amplite™ Ethanol Reagent (Component A) to make 250X Amplite™ Ethanol Reagent stock solution. The stock solution should be used promptly. Note:  The Amplite™ Ethanol Reagent is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Ethanol Reagent is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH 7 – 8. The provided assay buffer (pH 7.4) is recommended.

2. Ethanol Enzyme Mix (100X):
Add 100 µL of Assay Buffer (Component B) into the vial of Ethanol Enzyme Mix (Component C) and mix well to make 100X Ethanol Enzyme Mix.


Ethanol standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/40001

Prepare an Ethanol standard by diluting the appropriate amount of the 100% Ethanol Standard (Component E) into H2O to produce Ethanol concentration ranging from 0% to 0.1%. A 0% Ethanol control is included as blank control. The final Ethanol concentrations should be two folds lower (i.e., 0% to 0.05%).


Add 20 μL of 250X Amplite™ Ethanol Reagent Stock Solution and 50 μL of 100X Ethanol Enzyme Mix into 5 mL of Assay Buffer (Component B) to make Ethanol working solution.


Table 1. Layout of Ethanol standards and test samples in a solid black 96-well microplate. ES= Ethanol Standards (ES1 - ES7, 0.0001% to 0.1%), BL=Blank Control, TS=Test Samples. 


Table 2. Reagent composition for each well.

ES1 - ES750 µLSerial Dilutions (0.0001% to 0.1%)
BL50 µLH2O
TS50 µLtest sample
  1. Prepare Ethanol standards (ES), 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: High concentration of Ethanol (e.g. 0.5% final concentration) may cause reduced fluorescence signal due to the over oxidation of Amplite™ ethanol reagent (to a non-fluorescent product).

  2. Add 50 µL of Ethanol working solution to each well of Ethanol standard, blank control, and test samples to make the total Ethanol assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Ethanol working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 5 to 30 minutes, protected from light.

  4. Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm), Cutoff = 570 nm. Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading.


Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)571
Emission (nm)584



View all 7 citations: Citation Explorer
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Novel Role of Ethanol in Delaying Postharvest Physiological Deterioration and Keeping Quality in Cassava
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Journal: Food and Bioprocess Technology (2019): 1--10
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Authors: Suwannoi, Panita and Chomnawang, Mullika and Sarisuta, Narong and Reichl, Stephan and M{\"u}ller-Goymann, Christel C
Journal: Journal of Ocular Pharmacology and Therapeutics (2017): 743--752
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Authors: Suwannoi, Panita and Chomnawang, Mullika and Sarisuta, Narong and Reichl, Stephan and Müller-Goymann, Christel C
Journal: Journal of Ocular Pharmacology and Therapeutics (2017)
Enhancement of the antioxidant and starch hydrolase inhibitory activities of king coconut water (Cocos nucifera var. aurantiaca) by fermentation with kombucha 'tea fungus'
Authors: Watawana, Mindani I and Jayawardena, Nilakshi and Gunawardhana, Chaminie B and Waisundara, Viduranga Y
Journal: International Journal of Food Science & Technology (2016): 490--498
On the dynamics of kefir volatome
Authors: Hu, Jie-Bi and Gunathilake, Sampath and Chen, Yu-Chie and Urban, Pawel L
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View all 3 references: Citation Explorer
Prevalence of ethanol consumption may be higher in women than men in a university health service population as determined by a biochemical marker: whole blood-associated acetaldehyde above the 99th percentile for teetotalers
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Journal: J Addict Dis (1998): 13
Quantifying ethanol by high performance liquid chromatography with precolumn enzymatic conversion and derivatization with fluorimetric detection
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