Microtubule preparation

Porcine tubulin was polymerized in BRB80 buffer (80 mM piperazine-N,N′-bis[2-ethanesulfonic acid], 1 mM EGTA, 1 mM MgCl₂ (pH 6.9)) with 5% dimethyl sulfoxide, 4 mM MgCl₂, and 1 mM GTP for 1.5–2 h at 37°C. Afterwards, microtubules were diluted with BRB80 containing 10 μM taxol (BRB80T), spun down in an Airfuge (Beckman Coulter, Brea, CA), and resuspended in BRB80T. If not noted otherwise, all chemicals are from Sigma (St. Louis, MO). Microtubules were visualized with differential interference contrast employing a light-emitting diode (26).