Exercise 6.10 - Myelin Lipid Extraction
Fraction A: Cholesterol
- To the purified myelin vessicles, add 4 volumes of acetone.
Blend the mixture for 1 minute at high speed.
- Add the blended mixture to a beaker and manually continue
stirring for 5 minutes.
Do not use any electric stirring motors.
Filter the mixture using a Buchner funnel and cold trap
attached to a water aspirator for reduced pressure. Save the
filtered acetone solution.
- Wash the residue from the filter paper back into the
blender. Add 100 ml. of fresh acetone and reblend for 1 minute.
- Add the second homogenate to the beaker, stir for 5 minutes
Combine the two filtered acetone portions.
- The acetone extracter material is FRACTION A. It contains
Before storing, it should be dried to remove the acetone.
This is best accomplished by flash evaporation or lyophilization.
Alternatively, the solution can be allowed to evaporate in a fume
hood designed to vent organic solvents. There will, of course, be
some decomposition with the latter technique. Evaporate the
acetone, collect the dry crude cholesterol and obtain its dry
- Keep the acetone insoluble residue (on the filter paper) for
extraction of Fractions B and C.
Fraction B: Lecithin and Cephalin
- Residue from extraction of Fraction A
- Petroleum ether
- Buchner funnel
- Extract the acetone insoluble material left over from Step 6
of the extraction of Fraction A with 200 ml of ether. Gently stir
for 5 minutes.
- Collect the filtrate using a Buchner Funnel, as above.
- Place the ether insoluble material on the filter into a
flask and re-extract with an additional 200 ml of ether. Repeat
the filtration and re-extraction one final time with a third 200
ml of ether.
- Combine all three ether filtrates.
Save the residue for Fraction C.
- Evaporate the ether filtrates to 50 ml under reduced
pressure. Pour the concentrated extract into 200 ml of acetone
- Collect the precipitate on filter paper and discard the
filtrate. The precipitate left on the filter paper FRACTION B,
containing lecithin and cephalin.
- Obtain the dry weight of Fraction B.
Fraction C: Sphingosine Phosphatides and Glycosides
- Residue from extraction of Fraction B
- Extract the ether insoluble residue obtained in the steps
above with 50 ml. of boiling ethanol. Vacuum filter the hot
ethanol while it is still hot. Discard the residue.
Use steam heat only and work in a laboratory properly equipped
for organic extractions. Hot ethanol has a very low flash point -
it will explode!
- Cool the ethanol filtrate and collect the precipitate by
vacuum filtration. This precipitate is Fraction C.
- Obtain the dry weight of Fraction C.
- Determine the percentage of each Fraction dry weight
relative to the original wet weight of calf brain.
Dissolve each of the Fractions to a final concentration of
1% (w/v) in chloroform:methanol (3:1) before further analysis.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com