For the present study, we used commercial bovine brain tubulin (catalog No T238, Cytoskeleton, Denver, CO, USA). Tubulin was polymerized with a “hybrid” protocol in either the presence or absence of Paclitaxel (Taxol Equivalent, Invitrogen™, P3456), following general guidelines from the Mitchinson Lab’s online protocols (http://mitchison.med.harvard.edu/protocols/ ). Briefly, all reactions were conducted in BRB80 solution containing 80 mM PIPES (1,4-piperazinediethanesulfonic acid), 1 mM MgCl2, 1 mM EGTA, and pH 6.8 with KOH. MT sheets were obtained as previously reported 7 (link).
In the experiments where MTs were polymerized in the presence of Paclitaxel (stock solution, 10 mM), the drug was initially prepared as per the manufacturer's recommendation and diluted to obtain solutions between 2 and 200 mM, which were added sequentially every 5 min to the tubulin suspension as a dilution 1:10 in BRB80, DTT and GTP.
In the experiments where MTs were polymerized in the presence of Paclitaxel (stock solution, 10 mM), the drug was initially prepared as per the manufacturer's recommendation and diluted to obtain solutions between 2 and 200 mM, which were added sequentially every 5 min to the tubulin suspension as a dilution 1:10 in BRB80, DTT and GTP.
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