CELL AND MOLECULAR BIOLOGY MANUAL 10

Experiment No. 5: Chemical Composition of Myelin

Myelin is an electrically-insulating dielectric phospholipid layer that
surrounds only the axons of many neurons. It is an outgrowth glial cell: Schwann cells
supply the myelin for peripheral neurons, whereas oligodendrocytes supply it to those
of the central nervous system. Myelin is considered a defining characteristic of the
(gnathostome) vertebrates, but it has also arisen by parallel evolution in some
invertebrates. Myelin was discovered in 1854 by Rudolf Virchow.

Myelin made by different cell types varies in chemical composition and

configuration, but performs the same insulating function. Myelinated axons are white
in appearance, hence the "white matter" of the brain.

Myelin is composed of about 80% lipid and about 20% protein. Some of the
proteins that make up myelin are Myelin basic protein (MBP), Myelin
oligodendrocyte glycoprotein (MOG), and Proteolipid protein (PLP). Myelin is made
up primarily of a glycolipid called galactocerebroside. The intertwining of the
hydrocarbon chains of sphingomyelin serve to strengthen the myelin sheath.

MATERIALS: calf brain, 0.25 M sucrose, 0.88 M sucrose, Sudan III dye, blender,
refrigerated centrifuge, 1.7 ml eppendorf tubes

PROCEDURE:

A. Isolation

1. Using a blender, homogenize calf brain in 0.25 M sucrose (100g in 90ml).

2. Centrifuge the homogenate at 2000 rpm for 10 minutes at 4C.
3. Collect the supernatant and centrifuge at 13,000 rpm for 10 minutes.
4. With a flick of the wrist, decant the supernate and resuspend the pellet in 10l
0.25 M sucrose.
5. Using a micropipettor, carefully add 25l 0.88 M sucrose so that the pellet
floats on top of the diluent.
6. Centrifuge at 13,000 rpm for 10 minutes.
7. Using a micropipettor, carefully collect the upper portion of the supernate.
This contains the myelin while the pellet contains the mitochondria.
8. Add several drops of Sudan III dye to the tube containing the supernate.
9. Observe your results.

B. Critical Thinking Questions:

1. What happened to the isolated myelin solution when Sudan III dye was added?
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2. Describe a positive Sudan III test.

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3. What are the limitations of this test?

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4. What are the functions of myelin?

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