Exercise 9.4 - Isolation of Microtubules (Bovine Brain)
- 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
- Bradford protein assay
- 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.
- 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.
- Centrifuge the crude brain homogenate at 19,600 xg for 30 minutes at 4° C to remove connective tissue and cellular debris.
- Decant the supernatant and recentrifuge the supernatant at 27,000 xg for 45 minutes at 4° C for further clarification.
- 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.
- Decant the remainder of the crude supernatant into a chilled beaker, and add an equal volume of 8 M glycerol in MT buffer.
- 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.
- 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.
- 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.
- With each successive polymerization with GTP and subsequent collection of the precipitated microtubules, repeat the protein determination on each sample.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org