Microtubule Co-Pelleting Assay
At relatively high centripetal forces, polymerized microtubules will sediment while unpolymerized tubulin protein will remain suspended in solution. This biophysical property can be leveraged in probing for microtubule-associated proteins and assessing tubulin polymerization competency (when stabilizing factors such as taxol are excluded). This protocol outlines how to fractionate tubulin protein solutions by centrifugation and probe the resulting pellet and supernatant fractions.
Assemble the polymerization mix on ice.
- 34 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
- 5 µl tubulin protein @ 20 mg/ml | [final] = 2 mg/ml
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 90,000 rpm (350,000 x g) for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100). Collect the supernatant on ice in a clean microtube.
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 @ 2 µM. Incubate for 10 minutes.
- Add a 1:10 volume of taxol @ 20 µM. Incubate for 10 minutes.
- Add a 1:10 volume of taxol @ 200 µM. Incubate for 15 minutes.
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. 20 µM). Taxol must be included in all subsequent buffers.
6. Assemble Reaction
Mix the protein of interest with the taxol-stabilized microtubules at 1 μM each in 100 μl reaction buffer (10 mM HEPES, pH=7.7, 50 mM KCl, 1 mM DTT, 20 μM taxol).
After polymerizing, taxol-stabilized microtubules are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULES ON ICE!
7. Incubate Reaction
Incubate the reaction at room temperature for 15 minutes.
Layer the reaction over a 150 ul sucrose cushion (10 mM HEPES, pH 7.7, 50 mM KCl, 20 μM taxol, 40% sucrose w/v) in an ultracentrifuge rotor (i.e. TLA 100). Spin at 60,000 rpm (150,000 x g) for 20 minutes at 26°C.
9. Collect Fractions
- Collect the supernatant fraction and add an equal volume of 2X Sample Buffer.
- Gently wash the supernatant/cushion interface twice with reaction buffer.
- Pipette off and discard the cushion.
- Gently wash the pellet twice with reaction buffer.
- Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.
Analyze the pellet and supernatant fractions by SDS-PAGE. Polymerization reactions devoid of GTP can serve as a negative control.