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American Society for Cell Biology, Molecular Biology of the Cell, 22(23), p. 4393-4401

DOI: 10.1091/mbc.e12-06-0444

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One-step purification of assembly-competent tubulin from diverse eukaryotic sources

Journal article published in 2012 by Per O. Widlund ORCID, Marija Podolski, Simone Reber, Joshua Alper, Marko Storch, Anthony A. Hyman ORCID, Jonathon Howard, David N. Drechsel
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from S. cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule associated proteins (MAPs) and motor proteins, which has been a major caveat in microtubule related research.