Ethanol Quantification assay
Marcos Valenzuela-Ortega
Abstract
Chemoenzymatic method for quantification of ethanol with a spectrophotometer at 500 nm (not UV !)
Based on Lewicka 2014 (DOI: 10.1021/sb500020g )
Adapted for analysis in plate reader of multiple samples at the same time.
Steps
Prepare culture samples
Sample your culture and spin it 16000rpm.
Transfer Supernatant to new tube and keep On ice if analysed inmediately, or freeze if analysed in a different day.
Prepare stocks
Prepare the following stocks using your tris buffer:
| A | B | C | D |
|---|---|---|---|
| Stock | mg/mL | mM | Mw |
| Yeast Adh | 5 | ||
| NAD+ | 13.3 | 20 | 685.41 |
| PMS | 6.1 | 20 | 306.34 |
| INTV | 25.3 | 50 | 505.00 |
| Ethanol | 46 (36.3 uL/mL) | 1000 | 46 |
INTV needs to be dissolved in buffer-DMSO (50:50)
Keep reagentsOn ice
(no need for ethanol)
Keep the buffer at Room temperature!
Prepare your ethanol standard ladder by making different dilutions of your 1M ethanol with tris buffer. The standard could be, for example, 400-200-100-50-25-10-5-0 mM ethanol.
Prepare reaction mastermix
Prepare reaction mix for all your samples.
Calculate the volume to be prepared should be 200 µL * well * 1.1 (so you have)
When counting "wells", consider replicates for samples and standards.
That is the volum of Tris you need, to which you have to add:
- Yeast ADH: 5 µL per mL
- NAD+: 1µL per mL
- PMS: 1 µL per mL
- INTV: 20 µL per mL
Reaction plate and incubation
Plan how to load samples in plate, and annotate it.
Consider that the reaction mix should be loaded to the replicates at different moments (so their variability comprises the variability caused by loading the mix at the different times.)
Load 200 µL of buffer to the blank wells. (some replicates here are good too). These wells wont receive reaction mix.
Load 10 µL of sample to the well bottom. Be sure all the volume of liquid is in the well and not in your pipette tip.
Load 200 µL of reaction mix to the wells with your multichannel pipette with as little difference of time between sample as possible.
Mix & incubate
Mix the plate (the plate reader can do this).
Incubate the plate for Room temperature for 15 minutes, in the dark.
Measure Abs 500 nm in plate reader at Room temperature
Analysis and troubleshooting
Blank should be done with water, samples measurements will be reliable only in the linear region of the standard.
BMG plate readers do most of the analysis for you, if you tell the software which wells had each blank, standarda and sample. You can introduce this in the software before or after the readings.
You can have a positive control (negative control culture spiked with ethanol).
Sensitivity can be increased using 20 µL of sample in 200 µL of mastermix, or increasing incubation time to 30 minutes.
