title: Isolation of Microtubules and Microtubule-Associated Proteins.
abstract: Microtubules are essential cellular structures in plant cells. They are polymerized from tubulin dimers and are regulated by microtubule-associated proteins (MAPs). Here, we describe a protocol for purifying tubulin dimers and MAPs from plant cells. The protocol involves preparing vacuole-free mini-protoplasts, a high quality cytoplasmic extract, cycles of microtubule polymerization and depolymerization to increase tubulin and MAP concentration, separation of tubulin and MAPs by column chromatography. We also present tubulin purification methods for biochemical assays.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro, Tokyo, 153-8902, Japan. hama.micro@bio.c.u-tokyo.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hamada
authors.affiliation: Department of Life Sciences, Graduate School of Life Sciences, University of Hyogo, Hyogo, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Sonobe
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D018929:Cell Culture Techniques; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D002852:Chromatography, Ion Exchange; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008870:Microtubules; D059828:Plant Cells / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011205:Povidone / Q000737:chemistry; D055503:Protein Multimerization; D011523:Protoplasts / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry; D014026:Nicotiana / Q000737:chemistry*; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D003470:Culture Media; D008869:Microtubule-Associated Proteins; D010940:Plant Proteins; D014404:Tubulin; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone
keywords: Arabidopsis thaliana; Mini-protoplasts; Tobacco; Tubulin