Unlabeled porcine tubulin

Unlabeled porcine tubulin and its labeled analogs, (TRITC and biotin), GTP and Paclitaxel were purchased from Cytoskeleton, Inc. Mouse monoclonal anti-6xHis tag antibody and rat tubulin antibody which recognizes the C-terminal tail of α tubulin were purchased from ABCAM. GMPCPP was purchased from Jena Biosciences, Germany. Streptavidin-coated polystyrene beads 1% w/v (0.82 μm in diameter) and silica microspheres 9,92 % solid w/v (5.0 μm in diameter) were purchased from Spherotech. Amyl acetate and 2% Colloidon in Amyl acetate were purchased from Electron Microscopy Sciences. Glass coverslips 22 × 45 × 1.5 mM were purchased from Fisher Scientific. Glucose oxidase from Aspergillous niger, aqueous solution of catalase from bovine liver, dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), ATP, MgCl_2, and Subtilisin A Bacillus licheniformis were purchased from Sigma Aldrich. Mouse anti-tubulin b3 antibody which recognizes the C-terminal tail of β tubulin was purchased from Bio-Rad Laboratories. Except where noted, all experiments were carried out in BRB80 buffer (80 mM PIPES, 1 mM MgCl₂, 1mM EGTA, 2 mM MgATP, pH 6.9, 184 mM ionic strength). A subset of single-molecule TIRF measurements were carried out in BRB12 buffer (12 mM PIPES, 1 mM MgCl₂, 1mM EGTA, 2 mM MgATP, pH 6.9, 36 mM ionic strength). Detailed ionic strength calculations are included in SI Appendix.