Feb 01, 2022
Version 2
Total Lipid Extraction from Baker's yeast (Saccharomyces cerevisiae) V.2
- Israel Olayide1,
- Monica Rieth2
- 1St. Louis University Dept. of Chemistry;
- 2Southern Illinois University-Edwardsville
- Labyrieth
Protocol Citation: Israel Olayide, Monica Rieth 2022. Total Lipid Extraction from Baker's yeast (Saccharomyces cerevisiae). protocols.io https://dx.doi.org/10.17504/protocols.io.b4h8qt9wVersion created by Monica Rieth
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 01, 2022
Last Modified: February 01, 2022
Protocol Integer ID: 57632
Keywords: lipids, extraction,
Abstract
This protocol outlines a method for extracting total lipids from Baker's yeast, Saccharomyces cerevisiae. It has been adapted from Roy et al., J. Lipid Res. 2018. doi: 10.1194/jlr.M088559.
Attachments
Guidelines
All culture ODs should be normalized for comparative analyses prior to the extraction. Weighed Eppendorf tubes to be used before getting started.
Extracted lipids can be stored at -20 degC
Safety warnings
chloroform and methanol should be used in a chemical hood
Day 1-3
Day 1-3
3d
3d
Sterilize the inoculating loop by dipping it in the ethanol and heating it for 30 second in the flame
Allow loop to cool the loop at least for 10 seconds
Streak plate from prepared glycerol stock (-80 °C) and spread on YPD agar plate
Allow plate to incubate for 2-3 days at 25-30 °C until colonies are 1-2mm in diameter
Day 4
Day 4
1d
1d
Inoculate 5 mL YPD with yeast colony from plate
Grow overnight at 30°C with shaking at 250 rpm
Day 5
Day 5
1d
1d
Check OD600 at UV-Vis spectrophotometer
Harvest the whole cells when OD600 is 1.5
Centrifuge at 1000 x g for 2 minutes or until clear to pellet the yeast
Pour off the supernatant without disturbing cell pellet
Wash the cells three times by resuspending pellets in 1ml PBS, centrifuge for at 500 x g for 3-5 mins.
After washing three times with PBS, resuspend in 1ml PBS and transfer this mixture to a 15 ml conical centrifuge tube.
Add 3.75 mL of chloroform/methanol (1:2) to the 1 mL of resuspended cells
Add 1.25 mL chloroform and 1.25 mL sterile water subsequently.
Vortex vigorously for 5 mins, and centrifuge at 150 x g for 5 mins at room temperature.
After centrifugation, a two-phase system is obtained: aqueous top phase and organic bottom phase which contains the lipids.
Carefully remove the top aqueous layer and the middle insoluble layers (precipitated proteins).
The organic bottom layer is dried using a speed vacuum or dried under a steady stream of nitrogen.
Record the weight of the dried sample by pre-weighing an Eppendorf tube or equivalent before or after drying the lipid.