NOTE: THE ASSAY IS VERY SENSITIVE TO ALCOHOL VAPOR. EXTREME CARE SHOULD BE TAKEN TO ENSURE THAT NO ALCOHOL VAPORS (ETHANOL, METHANOL, PROPANOL) ARE IN THE LABORATORY AIR WHERE THE ASSAY IS TO BE PERFORMED. LABORATORIES WHERE HPLC EQUIPMENT AND SOLVENTS ARE STANDING OR WHERE ALCOHOL IS USED TO WIPE DOWN LABORATORY BENCHES OR EQUIPMENT ARE INAPPROPRIATE LOCATIONS TO PERFORM THIS ASSAY.
1. Standard Curve Preparations: For the colorimetric assay, add 50 µL of pure ethanol standard (4 °C, ice cold) to 821.4 µL Ethanol Assay Buffer, mix well. Then take 10 µL of the dilution into 990 µL assay buffer to generate 10 nM/µL of ethanol standard. Take 100 µL of the dilution into 900 µL assay buffer to generate 1 mM (1nM/µL). Add 0, 2, 4, 6, 8, 10 µL to a series of wells in a 96 well plate and adjust the volume of each to 50 µL with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nM/well ethanol Standard. Note: The fluorometric assay is 10-fold more sensitive than the colorimetric assay. For fluorometric measurement, dilute the ethanol standard to 0.1 mM by adding 100 µL of the ethanol standard to 900 µL of Reaction Buffer, mix well. Then add 0, 2, 4, 6, 8, 10 µL to a series of wells and adjust volume of each to 50 µL with Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nM/well ethanol Standard.
2. Sample Preparation: Samples can be diluted directly in Assay Buffer and tested. Biological samples such as serum (containing approx. 0.01-0.16 % w/v) should be diluted 1:10-1:100 and volumes in the range of 10-30 µL used. For beverages which contain 100X more alcohol, correspondingly greater dilutions should be used. We suggest making several dilutions of your sample so that the sample reading is within the standard curve range. When too much ethanol is in the reaction, the reaction develops from pink color to purple to brown, then becomes colorless.
3. Reaction Mix Preparation: Mix enough reagent for the number of assays performed: For each well, prepare a total 50 µL Reaction Mix containing: 46 µL Ethanol Assay Buffer 2 µL Ethanol Probe* 2 µL Ethanol Enzyme Mix *