title: One-step purification of assembly-competent tubulin from diverse eukaryotic sources.
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 Saccharomyces 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 and motor proteins, which has been a major issue in microtubule-related research.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
authors.forename: Per O
authors.initials: PO
authors.lastname: Widlund
authors.forename: Marija
authors.initials: M
authors.lastname: Podolski
authors.forename: Simone
authors.initials: S
authors.lastname: Reber
authors.forename: Joshua
authors.initials: J
authors.lastname: Alper
authors.forename: Marko
authors.initials: M
authors.lastname: Storch
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Hyman
authors.forename: Jonathon
authors.initials: J
authors.lastname: Howard
authors.forename: David N
authors.initials: DN
authors.lastname: Drechsel
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D016825:Chlamydomonas reinhardtii; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D018411:Spodoptera / Q000378:metabolism*; D014404:Tubulin / Q000302:isolation & purification*; D014982:Xenopus laevis
chemical_list: D008869:Microtubule-Associated Proteins; C109414:STU2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin