Exercise 9.4 - Isolation of Microtubules (Bovine Brain)
Procedure 3    1. Remove the meninges and peripheral blood
vessels from the brain. Puree the brain by pushing it through a
wire sieve and directly into a chilled beaker.    2. Homogenize
the cerebral hemispheres of the brain at 4° C in a tissue
homogenizer capable of homogenizing with minimal sheer force. 4
Use the maximum speed allowable for your homogenizer for 10
seconds. Place the brain hemispheres in the homogenizer with
microtubule buffer at a ratio of 0.5 ml of MT buffer to 1 g of
wet weight of brain tissue.    3. Centrifuge the crude brain
homogenate at 19,600 xg for 30 minutes at 4° C to remove
connective tissue and cellular debris.    4. Decant the
supernatant and recentrifuge the supernatant at 27,000 xg for 45
minutes at 4° C for further clarification.    5. Collect 10 ml
of the supernatant and determine the protein content of your
sample using the Bradford protein assay (Appendix G). Use bovine
serum albumin or lysozyme for establishing a