Ethanol Assay Kit

(Catalog #K620-100; 100 assays; Store at -20^oC)

I. Introduction:

Alcohol (ethanol C₂H₅OH) is among the most widely consumed drinks. Low dose of alcohol consumption may help blood circulation, while heavy alcohol consumption may lead to various form of disease. Quantitative determination of alcohol finds applications in basic research, drug discovery, clinic studies and wine industry processes. The Ethanol Assay Kit provides a simple, rapid, and sensitive method for accurate quantification of ethanol concentration in many types of biological samples, such as serum, plasma, body fluid, food or growth media. Alcohol oxidase oxidizes ethanol to generate H₂O₂ that reacts with ethanol probe to generate color and fluorescence, which can easily be quantified by either colorimetric (spectrophotometry at l = 570 nm) or fluorometric (at Ex/Em = 535/587 nm) methods. The kit detects 0.4-40 ppm alcohol (~10-800 nM).

1. 
   

   Kit Contents

   
   

Components	Volume	Cap color	Part Number
Ethanol Assay Buffer Ethanol Probe (lyophilized) Dimethylsulfoxide (DMSO) Ethanol Enzyme Mix (lyophilized) Ethanol Standard (MW:46.07, 17.15N)	25 ml 1 vial 0.4 ml 1 vial 0.5 ml	WM Red Brown Green Yellow	K620-100-1 K620-100-2 K620-100-3 K620-100-4 K620-100-5

2. 
   

   Reagent Preparation:

   
   

Ethanol Probe: Dissolve the Ethanol Probe in 220 μl DMSO (provided) prior to use. Store at –20^oC, protect from light. Use within two months.

Ethanol Enzyme Mix: Dissolve the Ethanol Enzyme Mix in 220 μl Ethanol Assay Buffer prior to use. The enzyme lost activity easily. Aliquot and store at –80^oC. Use within two months.

3. 
   

   Ethanol Assay Protocol:

   
   

The following protocol describes assays in 100 μl per micro-plate well.

1. 
   

   Standard Curve Preparations:

   
   

For colorimetric assay, add 11.7μl (9.2 mg) of pure ethanol standard into 988.3μl Ethanol Assay Buffer, mix well. Then take 10 μl of the diluent into 990 μl assay buffer to generate 2 nmol/μl of ethanol standard. Add the diluted ethanol standard 0, 4, 8, 12, 16, 20 μl into individual wells. Adjust volume to 50μl/well with Assay Buffer to generate 0, 8, 16, 24, 32, 40 nmol/well ethanol Standard.

Note: Fluorometric assay is 10 folds more sensitive than colorimetric assay. For fluorometric assay, dilute the ethanol standard 10 fold more by adding 100 μl of the 2 nmol/μl ethanol Standard to 900 μl of Reaction Buffer, mix well. Then add 0, 4, 8, 12, 16, 20 μl into individual wells. Adjust volume to 50 μl/well with Assay Buffer to generate 0, 0.8, 1.6, 2.4, 3.2, 4.0 nmol/well ethanol Standard. Then follow the same protocol. Read fluorescence at Ex/Em = 535/590 nm.

2. 
   

   Sample Preparations: Prepare samples in duplicates with reaction buffer to total volume 50 μl/well. We suggest making several dilutions of your sample with Assay Buffer, so that the sample reading can be within the standard curve range.

   
   

 

 

 

 

3. 
   

   Reaction Mix Preparation: Mix enough reagent for the number of assays performed: For each well, prepare a total 50 μl Reaction Mix containing:

   
   


4. 
   

   μl Ethanol Assay Buffer

   
   

2 ml Ethanol Probe*

2 ml Ethanol Enzyme Mix

*Note: For fluorometric assay, use 0.2 μl Ethanol Probe per well to reduce the fluorescence background.

4. Add 50 μl of the Reaction Mix to each well containing the Ethanol Standard or test samples.

5. 
   

   Incubate the reaction for 60 minutes* at room temperature, protected from light.

   
   

*Note: The reaction is continuous, the incubation time will depend on the conditions. We suggest to read the plate at several time points. The top reading should be below 2. Reading may decrease after the reading reach over 2, which will effect the linearity of the standard curve. Keep your sample readings at the same time as the standard curve reading.

6. 
   

   Measure O.D. 570 nm for colorimetric assay or Ex/Em = 535/590 nm for fluorometric assay in a micro-plate reader.

   
   


7. 
   

   Correct background by subtracting the background value derived from the 0 ethanol control from all samples (The background reading can be very significant and must be subtracted from sample readings). Calculate the ethanol concentrations of the test samples from the standard curve you generated, multiplied by the dilution factor.

   
   

C = Sa/Sv nmol/ul or mM

Where: Sa is sample amount from the standard Curve (nmol).

Sv is sample volume added into the sample well (ul).

Ethanol molecular weight: 46.07 g.mol^-1.

 

 

 

 

 

 

Ethanol Standard Curve. Ethanol standard was performed as described in the kit instructions.