Alcoholic drinks are among the daily consumed beverages. Studies have shown heavy alcohol consumption may lead to various forms of liver diseases and to increased mortality rates. Quantitative determination of alcohol (ethanol, C2H5OH) finds applications in basic research, drug discovery, clinic studies and winery.||Simple, direct and automation-ready procedures for measuring ethanol concentration are very desirable. Ethanol assay kit is based on an improved dichromate method, in which dichromate is reduced by ethanol to a bluish chromic (Cr3+) product. The intensity of color, measured at 580 nm, is a direct measure of the alcohol concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples and exhibits high sensitivity.||Applications:|Ethanol determination in alcohol containing samples such as beverages (e.g. wine, beer) and yeast cultures. For samples containing less than 0.1% alcohol such as serum or plasma, Specific Ethanol Assay Kit is recommended.||Key Features:|Sensitive and accurate. Detection range 0.04–4% alcohol in 96-well plate assay.|Convenient and high-throughput. The procedure involves adding a single working reagent, incubation for 8 min, adding a Stop Reagent, and reading the optical density. Can be readily automated as a highthroughput 96-well plate assay for thousands of samples per day.|Versatility. Assays can be executed in 96-well plate or cuvet.||Kit Contents: (500 tests in 96-well plates)|Reagent A: 50ml Reagent B: 50ml|10% TCA: 50ml Standard: 2ml 10% (v/v) ethanol|Storage conditions. The kit is shipped at room temperature. Store reagents at room temperature and the ethanol standard at 4°C. Shelf life: 12 months after receipt.|Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|Procedure using 96-well plate:|1. Prepare 600ul 2% Premix by mixing 120ul 10% Standard and 480ul distilled water. Dilute standard as follows. Transfer 100ul standards and samples into wells of a clear bottom 96-well plate.|No Premix+H2O Vol (ul) Ethanol (%)|1 150ul+0ul 150 2.00|2 120ul+30ul 150 1.60|3 90ul+60ul 150 1.20|4 60ul+90ul 150 0.80|5 45ul+105ul 150 0.60|6 30ul+120ul 150 0.40|7 15ul+135ul 150 0.20|8 0ul+150ul 150 0|2. Add 100ul Reagent A quickly using a multi-channel pipettor. Tap plate lightly to mix.|3. Incubate 8 to 30 min at room temperature. The reagent color changes from yellow to visibly bluish in wells 1-4. Add 100ul Stop Reagent B quickly using a multi-channel pipettor. Tap plate to mix.|4. Read OD at 570-600nm (peak 580nm).||Procedure using cuvette:|1. Prepare 2%, 1%, 0.5% standards and use distilled water as blank control. Transfer 400ul diluted Standards and 400ul samples to 1.5-mL centrifuge tubes.|2. Add 400ul Reagent A quickly to each tube and vortex briefly to mix.|3. Incubate 8 to 30 min at room temperature. Add 400ul Reagent B quickly and mix briefly.|4. Transfer to cuvettes and read OD at 570-600nm (peak 580nm).|Note: for the cuvette assay, it is recommended that an interval be applied between additions, e.g., add Reagent A to Tube 1 and 1 min later to Tube 2 etc. After the incubation step is completed, add the Stop Reagent B to Tube 1 and 1 min later to Tube 2 etc. This will ensure identical incubation time between tubes.||Calculation:|Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard alcohol concentrations. Determine sample ethanol concentration from the standard curve.|Conversions: 1% (v/v) ethanol equals 170mM or 785mg/dL.||Materials Required, But Not Provided:|Pipeting (multi-channel) devices.|Procedure using 96-well plate:|Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.|Procedure using cuvette:|Centrifuge tubes, table centrifuge, cuvets and spectrophotometer.||General Considerations:|1. If sample contains glucose or glycerol, use Specific ethanol assay kit. For samples containing only sugars (e.g. glucose), use Saccharide Removal Kit to remove the interferents prior to assay with the Ethanol Kit.|2. This assay is based on a kinetic reaction. Addition of Reagent A and B (Stop reagent) should be quick and mixing should be brief but thorough.|3. Sample pretreatment. Proteinaceous samples, e.g. plasma, serum, culture media, should be deproteinated by adding 1 vol sample to 2 vol 10% TCA (provided). Pellet for 5 min at 14,000 rpm on a table centrifuge, carefully transfer supernatant for assay (n=3). Saliva and urine can be analyzed directly (n=1). For wines, dilute samples to approximately 1 to 2% prior to assay.