Amplite® Ethanol Quantitation Kit

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

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

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.

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.

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.

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. 0.05% 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.