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Taxol represents a unique class of tubulin inhibitors in that it stabilizes microtubules from depolymerization. It can therefore be utilized in a lab setting to protect microtubules from depolymerization at room temperature. While tubulin protein can be polymerized in the presence of taxol, this has been shown to alter the structure of the subsequent microtubules. We therefore recommend adding taxol to already polymerized microtubules to minimize these effects. Note that when using taxol-stabilized microtubules, taxol must be included in all subsequent buffers. As taxol is highly insoluble in aqueous solutions, it is often necessary to also include DMSO at ~10% to prevent taxol precipitation.
Taxol-stabilized microtubules are relatively long and flexible. To generate short, rigid microtubules, see Generating GMPCPP-Stabilized Microtubules.
Assemble the polymerization mix on ice. Add labeled tubulin if desired. Fluorescent dye-labeled tubulin is typically included at a 1:4 ratio with unlabeled tubulin, although this ratio should be adjusted based on experimental application and specific product labeling stoichiometry ([dye]/[tubulin]). As a benchmark, a final ratio of 1 labeled tubulin heterodimer per 4 unlabeled tubulin heterodimers results in uniform and bright fluorescent microtubules. Biotinylated tubulin is typically included at a 1:50 ratio with unlabeled tubulin.
Incubate the polymerization mix on ice for 5 minutes. This allows the tubulin protein to bind GTP.
Clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm (350,000 x g) for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100). Recover the supernatant on ice in a clean microtube.
Induce polymerization by incubating the polymerization mix in a 37°C water bath for 1 hour.
Layer the taxol-stabilized microtubules over a 150 µl pre-warmed glycerol cushion (40% w/v glycerol in 1X Tubulin PEM Buffer) in an ultracentrifuge rotor (i.e. TLA 100). Spin at 90k rpm (350,000 x g) for 5 minutes at 27°C.