Purification and Biophysical Analysis of Microtubule-Severing Enzymes In Vitro - Methods in Cell Biology - page 196

g for 10 min at 4 C in a micro
ultracentrifuge or airfuge to remove any aggregated tubulin.
3. Collect the supernatant and add 1 mM GTP.
4. Incubate at 37 C for 30 min to nucleate and polymerize the tubulin into filaments.
5. Add 40 m M Taxol.
6. Incubate at 37 C for 30 min to equilibrate Taxol.
7. Spin down the microtubules at 16,000

2. Clarify the tubulin by centrifuging at 366,000

g in a tabletop centrifuge for 10 min at

27 C.
8. Resuspend the pellet in PEM-100 with 40 m M Taxol.

13.1.2.1.2.2 GMPCPP microtubules
1. Mix unlabeled tubulin (5 mg/ml) with labeled tubulin at any percentage from 5%
to 20% labeling. Treat lyophilized tubulin as above.
2. Clarify the tubulin by centrifuging at 366,000 g for 10 min at 4 Cin

ultracentrifuge to remove any aggregated tubulin.
3. Collect the supernatant and add 1 mM GMPCPP.
4. Incubate at 37 C for 30 min to nucleate and polymerize the tubulin into filaments.
5. Spin down the microtubules at 16,000

g in a tabletop centrifuge for 10 min at

27 C.
6. Resuspend the pellet in PEM-100.

13.1.2.1.2.3 Polarity-marked microtubules. Polarity-marked microtubules are
created by growing GTP-tubulin microtubules from GMPCPP-microtubule seeds.
In order to differentiate the seed from the elongation, they must have a 10-fold dif-

ference in labeling intensity in the same color channel, or be labeled with different
fluorophores. We do not grow GMPCPP seeds with Taxol, but we do use it to sta-
bilize elongated extensions after polymerization.

1. For both the GMPCPP seeds and the GTP-tubulin elongation, combine the
labeled tubulin with the unlabeled to the desired percentages. We recommend
20% labeling for seed and 3% labeling for elongations, if using the same
fluorophore.
2. Assemble GMPCPP seeds as you made GMPCPP microtubules above. Clarify
the elongation tubulin and keep it on ice.
3. After incubating the GMPCPP tubulin at 37 C, shear the GMPCPP microtubules
by passing them through a Hamilton syringe (gauge 22S) three times to create
short seeds.
4. Add 1 mM GTP to the elongation tubulin at 5 mg/ml and leave on ice.
5. Incubate the elongation tubulin for 1 min at 37 C to bring the solution up in
temperature, but not to polymerize yet.
6. Add 2 m l of the GMPCPP seeds to the elongation tubulin and incubate at 37 C
for 20 min to polymerize GTP extensions from GMPCPP seeds.
7. Add Taxol to 40 m M final concentration to stabilize elongated microtubules. Use
this type of microtubule within 12 h to prevent end-to-end annealing and loss of
polarity marker.