5. Add Taxol Stepwise
Add taxol to the polymerized microtubules in the following stepwise manner. Continue incubating in a 37°C water bath:
- Add a 1:10 volume of taxol @ 5 µM in DMSO. Incubate for 10 minutes.
- Add a 1:10 volume of taxol @ 50 µM in DMSO. Incubate for 10 minutes.
- Add a 1:10 volume of taxol @ 500 µM in DMSO. Incubate for 15 minutes.
NOTE :
Taxol must be added stepwise in order to avoid precipitation. The final taxol concentration should match or be in equimolar excess to the tubulin protein concentration (i.e. 50 µM). 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.