PC-purified tubulin (2 - 6 mg/mL; 2 mg/mL = 20 µM)

DMSO

100 mM Mg·GTP

4 mM Taxol in DMSO

PM =	100 mM PIPES pH 6.8 2 mM EGTA 1 mM Mg2SO4

Procedure

1.	Assemble microtublues by incubating 2 - 6 mg/mL PC-purified tubulin + 1 mM Mg·GTP + 5% DMSO at 37°C for 30 - 60 min. Add taxol in PM buffer (prewarmed to 37°C) to adjust final concentration to 60 µM taxol and 20 µM tubulin. Incubate 5 min at 37°C.

2.	Centrifuge microtubules at 70,000 rpm for 20 min at 22°C in a tabletop ultracentrifuge. Carefully remove the supernatant and discard.

3.

Resuspend pelleted microtubules in taxol in PM (pre-warmed to 37°C) by pipetting gently using a cut-off yellow tip to give a final concentration of 50 µM taxol and 20 µM tubulin. This may require several minutes of pipetting to ensure complete resuspension of the pellet. Avoid bubbles while pipetting.

4.	GTP-depleted microtubules can be divided into 50 - 100 µL aliquots, flash-frozen in liquid nitrogen, and stored at -80°C. To thaw, incubate frozen microtubules for 30 - 60 min at 37°C.

Modified from Huang & Hackney (JBC 269, 16493-501 1994)