Exercise 6.9 - Chemical Composition of Myelin

LEVEL III

Materials

• Calf brain 10
• 0.25 M sucrose
• 0.88 M sucrose
• Blender or tissue homogenizer
• Refrigerated centrifuge

Procedure

1. Homogenize 100 gms of calf brain in 0.25 M sucrose to make a 10% brei.

2. Centrifuge the homogenate at 500 xg for 10 minutes at 4° C.

3. Collect the supernatant and recentrifuge the supernatant at 11,000 xg for 10 minutes. 11

4. Discard the supernatant and resuspend the pellet in 10 ml. of 0.25 M sucrose.

5. 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.

6. 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.

7. Carefully remove the upper myelin vessicle layer.

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