LEVEL II

Materials

• Freshly removed bovine brain 2
  
• Wire sieve (tea strainer)
  
• Microtubule buffer (MT buffer)
  
  • 0.1 M MES (2-(N-Morphilino)ethanesulfonic acid)
    
  • 1 mM EGTA (Ethylene Glycol-bis(-aminoethyl Ether) N,N,N',N'-tetraacetic acid)
    
  • 0.5 mM MgCl
    
  • Adjust pH to 6.4
    
• 8 M Glycerol in MT buffer
  
• Virtis homogenizer (or equivalent)
  
• Refrigerated centrifuge
  
• GTP
  
• Bradford protein assay

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 standard curve.

6. Decant the remainder of the crude supernatant into a chilled beaker, and add an equal volume of 8 M glycerol in MT buffer.

7. Add dry GTP (MW 523) to the crude supernatant to make a 1.0 mM final concentration of GTP and incubate the mixture at 37° C for 30 minutes.

8. Centrifuge the mixture at 100,000 xg for 60 minutes at 25° C. It is crucial that the temperature be maintained at 25° C. At a lower temperature the microtubules will not polymerize (thus no pellet); and at a higher temperature the tubulin may be degraded.

9. Remove the pellet and resuspend in 40 ml of cold MT buffer. Incubate for 30 minutes at 4° C. Occasional homogenization in a ground glass homogenizer will facilitate depolymerization of the microtubules.

   For good tubulin polymerization, a series of polymerizations and depolymerizations, with subsequent centrifuge collections is required. Steps 7 through 9 should be repeated a minimum of 3 times.

10. With each successive polymerization with GTP and subsequent collection of the precipitated microtubules, repeat the protein determination on each sample.