Isolation of Microtubules (Bovine Brain)

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.