Exercise 6.9 - Chemical Composition of Myelin
- Calf brain
- 0.25 M sucrose
- 0.88 M sucrose
- Blender or tissue homogenizer
- Refrigerated centrifuge
- Homogenize 100 gms of calf brain in 0.25 M sucrose to make a
- Centrifuge the homogenate at 500 xg for 10 minutes at 4° C.
- Collect the supernatant and recentrifuge the supernatant at
11,000 xg for 10 minutes.
- Discard the supernatant and resuspend the pellet in 10 ml.
of 0.25 M sucrose.
- Carefully add 25 ml of 0.88 M sucrose to the bottom of the
tube, so that the resuspended pellet lies on top of the sucrose.
- Centrifuge at 11,000 xg for 10 minutes to separate the
mitochondria from the myelin vessicles. The myelin will float on
the surface, while the denser mitochondria should pellet at the
bottom of the tube.
- Carefully remove the upper myelin vessicle layer.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org