Exercise 6.5 - Extraction of Brain Lipids

LEVEL II

Materials

• Calf brain 4
• Chloroform
• Methanol
• 0.1 N HCl
• Blender or tissue homogenizer
• Refrigerated preparative centrifuge
• Large bucket rotor
• Freeze dryer
• Buchner funnel and filter paper (with cold trap)

Procedure

1. 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!

2. Centrifuge the mixture in a large scale rotor @ 10,000 xg for five minutes at 4 ° C to clarify the solution.

3. Add 20 ml. of distilled water per 100 ml of solution from step 2 and shake to form an emulsion.

4. Lyophilize to dryness.

5. 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.

6. Add 1.2 volumes of methanol.

7. Add 1.5 volumes of 0.1 N HCl and shake vigorously.

8. Centrifuge and collect the lower phase. Discard supernatant and interphase material.

9. Filter the lower phase three times with fresh filter paper.

10. 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.

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