Abstract
Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availability of purified αβ-tubulin. In this chapter, we describe the process of purification of heterodimeric αβ-tubulin from porcine brain.
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References
Horio, T. and Hotani, H. (1986), Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature 321: 605–7.
Mitchison, T. and Kirschner, M. (1984), Dynamic instability of microtubule growth. Nature 312: 237–42.
Howard, J. and Hyman, A. A. (2009), Growth, fluctuation and switching at microtubule plus ends. Nat Rev Mol Cell Bio 10: 569–74.
Desai, A. and Mitchison, T. J. (1997), Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 13: 83–117.
Howard, J. and Hyman, A. A. (2003), Dynamics and mechanics of the microtubule plus end. Nature 422: 753–8.
Brouhard, G. J., Stear, J. H., et al. (2008), XMAP215 is a processive microtubule polymerase. Cell 132: 79–88.
Helenius, J., Brouhard, G., et al. (2006), The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends. Nature 441: 115–9.
Akhmanova, A. and Steinmetz, M. O. (2008), Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat Rev Mol Cell Biol 9: 309–22.
Borisy, G. G., Marcum, J. M., et al. (1975), Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. Ann N Y Acad Sci 253: 107–32.
Castoldi, M. and Popov, A. V. (2003), Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32: 83–8.
Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973), Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci USA 70: 765–8.
Burton, K. (1969), Spectral data and pK values for purines, pyrimidines, nucleosides, and nucleotides., in: Data for Biochemical Research, R.M.C. Dawson, et al., Editors., Oxford University Press: Oxford and New York.
Mitchison Lab: “Large Scale Tubulin Preparation”. (http://mitchison.med.harvard.edu/protocols/tubprep.html).
Bradford, M. M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–54.
Acknowledgements
The authors acknowledge the input of many people in the development of these protocols. Protocols published on the Web, especially by the Mitchison lab, have proved invaluable in the development of this protocol. In more recent years, many members, past and present, of the Howard, Diez, and Hyman labs at the MPI-CBG have been involved in performing this purification. In particular, we thank Regine Hartmann, Corina Braeuer, Marija Zanic, Gero Fink, Tony Ashford, and Andy Hunter. Finally, C.G. and C.T.F. thank Gary Brouhard, both for sharing the misery of getting this protocol up and running (again) and for promising (after “the incident”) never to run another PC column.
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Gell, C., Friel, C.T., Borgonovo, B., Drechsel, D.N., Hyman, A.A., Howard, J. (2011). Purification of Tubulin from Porcine Brain. In: Straube, A. (eds) Microtubule Dynamics. Methods in Molecular Biology, vol 777. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-252-6_2
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DOI: https://doi.org/10.1007/978-1-61779-252-6_2
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