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Generating Taxol-stabilized Microtubules
Overview
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.
Protocols
1. Assemble
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.
- 26.5 µl H20 | final volume = 50 µl
- 10 µl Tubulin PEM Buffer @ 5X | [final] = 1X
- 0.5 µl DTT @ 100 mM | [final] = 1 mM
- 0.5 µl GTP @ 100 mM | [final] = 1 mM
- 12.5 µl tubulin protein @ 20 mg/ml | [final] = 5 mg/ml
2. Incubate
Incubate the polymerization mix on ice for 5 minutes. This allows the tubulin protein to bind GTP.
3. Clarify
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.
4. Polymerize
Induce polymerization by incubating the polymerization mix in a 37°C water bath for 1 hour.
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.
6. Pellet
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.
7. Resuspend
- Pipette off and discard the supernatant.
- Gently wash the supernatant/cushion interface twice with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 50 µM taxol + 10% DMSO.
- Pipette off and discard the cushion.
- Gently rinse the pellet twice with prewarmed 1X Tubulin PEM Buffer + 1 mM DTT + 50 µM taxol + 10% DMSO.
- Resuspend the pelleted taxol-stabilized microtubules with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 50 µM taxol + 10% DMSO to the original volume (i.e. 50 µl). Use a cut pipette tip to avoid shearing the microtubules.
NOTE :
After polymerizing, taxol-stabilized microtubules are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULES ON ICE!
The taxol-stabilized microtubules are now ready for experimental use and can be kept at room temperature for several days. Microscopy applications often require further diluting the microtubules in 1X Tubulin PEM Buffer + 1 mM DTT + 50 µM taxol + 10% DMSO until the desired density is achieved.
After polymerizing, taxol-stabilized microtubules are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULES ON ICE!
The taxol-stabilized microtubules are now ready for experimental use and can be kept at room temperature for several days. Microscopy applications often require further diluting the microtubules in 1X Tubulin PEM Buffer + 1 mM DTT + 50 µM taxol + 10% DMSO until the desired density is achieved.
