Ethanol Assay Kit

Catalog Number MAK076

Storage Temperature –20 °C

TECHNICAL BULLETIN

Product Description Precautions and Disclaimer

Ethanol is a psychoactive component of many This product is for R&D use only, not for drug,
commonly consumed drinks where it acts as a central household, or other uses. Please consult the Safety
nervous system depressant. After ingestion, ethanol is Data Sheet for information regarding hazards and safe
absorbed into the bloodstream via the stomach and handling practices.
small intestine. Ethanol is largely metabolized by the
liver but is also secreted in urine or through respiration. Preparation Instructions
The monitoring of ethanol levels is also important in Briefly centrifuge vials before opening. To maintain
fermentation processes. reagent integrity, avoid repeated freeze/thaw cycles.

The Ethanol Assay Kit provides a simple and reliable Ethanol Assay Buffer – Allow buffer to come to room
method for the quantification of ethanol in serum, temperature before use.
plasma, and other body fluids as well as in beverages
and growth media. Ethanol concentration is determined Ethanol Probe – Warm to room temperature to melt
by a coupled enzyme reaction, which results in a frozen solution prior to use. Aliquot and store
colorimetric (570 nm)/fluorometric (λex = 535/ protected from light and moisture at –20 °C. Upon
λem = 587 nm) product, proportional to the ethanol thawing, the Ethanol Probe is ready to use in the
present. colorimetric assay.

Components For the fluorescence assay, dilute an aliquot of the

The kit is sufficient for 100 assays in 96 well plates. Ethanol Probe Solution 5 to 10-fold with Ethanol
Assay Buffer, just prior to use. This will reduce the
Ethanol Assay Buffer 25 mL background of the fluorescence assay.
Catalog Number MAK076A
Ethanol Enzyme Mix – Reconstitute in 220 µL of
Ethanol Probe, in DMSO 0.2 mL Ethanol Assay Buffer. Mix well by pipetting, then
Catalog Number MAK076B store, protected from light, at 2–8 °C. Stable for
2 months.
Ethanol Enzyme Mix 1 vl
Catalog Number MAK076C Storage/Stability
The kit is shipped on wet ice and storage at –20 °C,
Ethanol Standard, 17.15 N 0.5 mL protected from light, is recommended.
Catalog Number MAK076D
Note: This kit is highly sensitive to the presence of
Reagents and Equipment Required but Not short-chain alcohols (ethanol, methanol, and propanol).
Provided. Storage of this kit in the vicinity of alcohol vapors can
• 96 well flat-bottom plate – It is recommended to use result in the uptake of the alcohols by kit components,
black plates with clear bottoms for fluorescence resulting in very high backgrounds.
assays and clear plates for colorimetric assays.
• Fluorescence or spectrophotometric multiwell plate
reader
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Procedure Assay Reaction

This assay should not be performed in areas where 1. Set up the Master Reaction Mix according to the
alcohol-containing solvents are stored or where scheme in Table 1. 50 µL of the Master Reaction
laboratory benches are wiped down with alcohol. Mix is required for each reaction (well).

All samples and standards should be run in duplicate. Table 1.

Reaction Mix
Ethanol Standards for Colorimetric Detection
Dilute 50 µL of the 17.15 N Ethanol Standard with Reagent Volume
808.7 µL of the Ethanol Assay Buffer to generate a Ethanol Assay Buffer 46 µL
1 µmole/µL standard. Dilute 10 µL of the 1 µmole/µL Ethanol Probe 2 µL
standard solution with 990 µL of ethanol assay buffer to Ethanol Enzyme Mix 2 µL
generate a 10 nmole/µL solution. Dilute 100 µL of the
10 nmole/µL solution with 900 µL to generate a 2. Add 50 µL of the Master Reaction Mix to each of
1 nmole/µL ethanol standard. Add 0, 2, 4, 6, 8, and the wells. Mix well using a horizontal shaker or by
10 µL of the 1 nmole/µL standard solution into a 96 well pipetting, and incubate the reaction for 30 minutes
plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well at 37 °C or 60 minutes at room temperature. Cover
standards. Add Ethanol Assay Buffer to each well to the plate tightly and protect the plate from light
bring the volume to 50 µL. during the incubation.
Ethanol Standards for Fluorometric Detection 3. For colorimetric assays, measure the absorbance
Generate a 10 nmole/µL ethanol standard solution as at 570 nm (A570). For fluorometric assays, measure
for the colorimetric assay. Dilute 10 µL of the fluorescence intensity (λex = 535/λem = 587 nm).
10 nmole/µL ethanol standard with 990 µL of the
Ethanol Assay Buffer to generate a 0.1 nmole/µL
standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the
0.1 nmole/µL standard solution into a 96 well plate,
generating 0 (blank), 0.2, 0.4, 0.6, 0.8, and
1.0 nmole/well standards. Add Ethanol Assay Buffer to
each well to bring the volume to 50 µL.

Sample Preparation
Samples should be diluted in Ethanol Assay Buffer.

Bring samples to a final volume of 50 µL with Ethanol

Assay Buffer.

For unknown samples, it is suggested to test several

sample dilutions to ensure the readings are within the
linear range of the standard curve.
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Results
Calculations Concentration of Ethanol
The background for the assays is the value obtained for
the 0 (blank) Ethanol Standard. Correct for the Sa/Sv = C
background by subtracting the 0 (blank) value from all
readings. Background values can be significant and Sa = Amount of ethanol in unknown sample (nmole)
must be subtracted from all readings. Use the values from standard curve
obtained from the appropriate ethanol standards to plot Sv = Sample volume (µL) added into the wells
a standard curve. C = Concentration of ethanol in sample
Note: A new standard curve must be set up each time
the assay is run. Ethanol molecular weight: 46.07 g/mole

Subtract the blank value from the sample reading to Sample Calculation
obtain the corrected measurement. Using the corrected Amount of ethanol (Sa) = 5.84 nmole
measurement, the amount of ethanol present in the (from standard curve)
sample may be determined from the standard curve. Sample volume (Sv) = 50 µL

Concentration of ethanol in sample

5.84 nmole/50 µL = 0.1168 nmole/µL

0.1168 nmole/µL × 46.07 ng/nmole= 5.38 ng/µL

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Troubleshooting Guide

Problem Possible Cause Suggested Solution

Cold assay buffer Assay Buffer must be at room temperature
Omission of step in procedure Refer and follow Technical Bulletin precisely
Plate reader at incorrect wavelength Check filter settings of instrument
Assay not working
For fluorescence assays, use black plates
Type of 96 well plate used with clear bottoms. For colorimetric assays,
use clear plates
Samples prepared in different buffer Use the Ethanol Assay Buffer
Samples used after multiple freeze-thaw Aliquot and freeze samples if samples will be
cycles used multiple times
Samples with erratic
Presence of interfering substance in the
readings If possible, dilute sample further
sample
Use of old or inappropriately stored Use fresh samples and store correctly until
samples use
Thaw all components completely and mix
Improperly thawed components
gently before use
Use of expired kit or improperly stored Check the expiration date and store the
Lower/higher reagents components appropriately
readings in samples Allowing the reagents to sit for extended Prepare fresh Master Reaction Mix before
and standards times on ice each use
Refer to Technical Bulletin and verify correct
Incorrect incubation times or temperatures
incubation times and temperatures
Incorrect volumes used Use calibrated pipettes and aliquot correctly
Thaw and resuspend all components before
Use of partially thawed components
preparing the reaction mix
Pipetting errors in preparation of standards Avoid pipetting small volumes
Prepare a Master Reaction Mix whenever
Pipetting errors in the Reaction Mix
possible
Non-linear standard Pipette gently against the wall of the plate
Air bubbles formed in well
curve well
Standard stock is at incorrect Refer to the standard dilution instructions in
concentration the Technical Bulletin
Recheck calculations after referring to
Calculation errors
Technical Bulletin
Substituting reagents from older kits/lots Use fresh components from the same kit
Samples measured at incorrect
Check the equipment and filter settings
wavelength
Unanticipated results Samples contain interfering substances If possible, dilute sample further
Sample readings above/below the linear Concentrate or dilute samples so readings
range are in the linear range

KVG,LS,MAM 04/15-1

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