title: Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts.
abstract: For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro.
authors.affiliation: Department of Systems Biology, Harvard Medical School, 536 Warren Alpert Building, 200 Longwood Avenue, Boston, MA, 02150, USA.
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Groen
authors.affiliation: Department of Systems Biology, Harvard Medical School, 536 Warren Alpert Building, 200 Longwood Avenue, Boston, MA, 02150, USA. timothy_mitchison@hms.harvard.edu.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Mitchison
mesh_terms: D000818:Animals; D002474:Cell-Free System; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D008941:Spindle Apparatus / Q000378:metabolism; D013194:Staining and Labeling; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*
chemical_list: D014404:Tubulin
keywords: Cell-free cytoplasm; Microtubules; Tubulin; Xenopus laevis