Lipid extraction for TLC (Chloroform-Methanol, Thiele lab)

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Lipid extraction for TLC (Chloroform-Methanol, Thiele lab)

This protocol describes extraction of lipids from cultivated cells for subsequent analysis by TLC. It can be adapted for extraction of tissue homogenates or any other biological material. It can also be scaled down or up for any size of tissue culture dish.


Step 1:
Grow cells in 10 cm dishes and perform the desired experimental manipulations. In the end, wash the cells three times with ice-cold PBS.

Step 2:
With a soft cell scraper (see below), scrape the cells into 1 ml cold PBS and pipet into 15 ml tubes (see below) containing 4 ml Methanol/Chloroform 2/1 and mix. You should have a homogenous single phase. Any cellular metabolism is now stopped.

Step 3:
Spin at 4000 rpm for 5 min, and transfer the supernatant into a new tube. The pellet consists mostly of proteins and can be discarded or kept for further analysis.

Step 4:
Add 1 ml of 50 mM citric acid, 2 ml of water and 1 ml of chloroform and shake. You will obtain a turbid 2-phase mixture.

Step 5:
Spin at 4000 rpm in a non-cooled centrifuge for 5-20 min. You will obtain a lower chloroform phase, an upper water/methanol phase, and an interphase that consists mostly of precipitated protein.

Step 6:
Discard the upper liquid (water/methanol) phase, leaving the protein layer untouched. Transfer the lower (chloroform) phase (without any droplet of the upper phase!!) into a new tube. Use glass pipettes or high-quality plastic throughout. For transfer of the chloroform phase, pre-rinse the pipette with chloroform two times.
This chloroform phase now contains all unpolar lipids (cholesterol and its esters, mono-, di- and triglycerides, waxes etc.) as well as most of the polar lipids (glycerophospholipids, sphingomyelin and the simple glycolipids up to GM3). Some complex glycolipids of the GM, GD, GT and globo series will be lost into the water/methanol phase. Also, phosphoinositides will not be completely recovered.

Step 7:
Evaporate the solvent completely in a stream of nitrogen. Dissolve in 30-50 µl of chloroform/methanol 2/1.

Soft cell scraper: Most commercial single-use cell scrapers are made from a relatively hard plastic, which works very poorly. To get a good, efficient scraper, take a stopper made from white silicon rubber (diameter 1-4 cm, according to the desired size of the scraper), and cut with a sharp knife a 2-3 mm thick round slice. This slice you cut into two half discs. Take care to get straight edges. This is a great cell scraper, which you can hold either with a paper clip or connect to a plastic pipette (by a hole that you punch at the round side) 

Tubes and tips: For TLC separation, followed by analysis by sulfuric acid charring, autoradiography or fluorescence detection, plastic tubes and tips made from high quality polypropylene are sufficient. For subsequent analysis by mass spectrometry, glassware (pre-rinsed with chloroform) should be used. Never use tissue culture plastic pipettes and other similar material from polystyrene or polycarbonate for organic solvents.

Chloroform: Chloroform can chemically decompose, driven by light and oxygen, into highly reactive compounds. In particular, HCl, phosgen and chlorine will be formed. In order to avoid this, chloroform is usually doted with a stabilizer such as ethanol or amylene. Particularly, amylene-stabilzed chloroform tends to cause problems. It is highly recommended to use ethanol-stabilized chloroform with a certificate for absence of phosgene and HCl, such as the one offered by Fluka (No. 25690)

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