Exercise 6.5 - Extraction of Brain Lipids
- Calf brain 4
- 0.1 N HCl
- Blender or tissue homogenizer
- Refrigerated preparative centrifuge
- Large bucket rotor
- Freeze dryer
- Buchner funnel and filter paper (with cold trap)
- Homogenize 25 g of white matter from calf brain, in 500 ml
of chloroform:methanol mixture (2:1, v/v).
Do not use an ordinary blender. The solvents will dissolve
the gasket and may burst into flames!
- Centrifuge the mixture in a large scale rotor @ 10,000 xg
for five minutes at 4 ° C to clarify the solution.
- Add 20 ml. of distilled water per 100 ml of solution from
step 2 and shake to form an emulsion.
- Lyophilize to dryness.
- Dissolve the residue in 50 ml of chloroform and filter
through three layers of filter paper in a buchner funnel. Use a
water vacuum system and a cold trap to prevent chloroform vapors
from entering a pump and causing a fire.
- Add 1.2 volumes of methanol.
- Add 1.5 volumes of 0.1 N HCl and shake vigorously.
- Centrifuge and collect the lower phase. Discard supernatant
and interphase material.
- Filter the lower phase three times with fresh filter paper.
- Add 1/3 volume of methanol to the filtrate and store in 1.0
ml lots in a freezer. The extract will remain stable for
approximately 2 months.
Return to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org