Microtubule Co-Pelleting Assay

Overview


At relatively high centripetal forces, polymerized microtubules will pellet while unpolymerized tubulin will remain suspended in solution. This biophysical property can be exploited in probing for microtubule-binding agents and assessing tubulin polymerization competency (when stabilizing factors such as Taxol are excluded).

For general tubulin handling instructions, please see “Storing and Handling Cryopreserved Tubulin” or “Storing and Handling Lyophilized Tubulin.”

Overview


At relatively high centripetal forces, polymerized microtubules will pellet while unpolymerized tubulin will remain suspended in solution. This biophysical property can be exploited in probing for microtubule-binding agents and assessing tubulin polymerization competency (when stabilizing factors such as Taxol are excluded).

For general tubulin handling instructions, please see “Storing and Handling Cryopreserved Tubulin” or “Storing and Handling Lyophilized Tubulin.”

Recommended Products


Unlabeled Tubulin

Unlabeled Tubulin

Buffer

Recommended Products


Recommended Products

Unlabeled Tubulin
Buffer

Protocol


Step 1

1. Assemble

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 @ 20 mg/ml | [final] = 2 mg/ml

Step 2

2. Incubate

Incubate the polymerization mix on ice for 5 minutes. This allows tubulin to bind GTP.

Step 3

3. Clarify

Clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm for 5 minutes at 4°C in a TLA-100 tabletop ultracentrifuge rotor.

Step 4

4. Polymerize

Induce polymerization by incubating the polymerization mix in a 37°C water bath for 1 hour.

Step 5

5. Add Taxol Stepwise

Add Taxol to the polymerized microtubules in the following stepwise manner. Continue incubating in a 37°C water bath:


  • Add 2 µM Taxol at a 1:10 volume ratio (i.e. 5 µl). Incubate for 10 minutes.
  • Add 20 µM Taxol at a 1:10 volume ratio (i.e. 5.5 µl). Incubate for 10 minutes.
  • Add 200 µM Taxol at a 1:10 volume ratio (i.e. 6 µl). 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 tubulin (i.e. 20 µM). Taxol must be included in all subsequent buffers.

Step 6

6. Assemble Reaction

Mix protein of interest with 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).

Step 7

7. Incubate Reaction

Incubate the reaction at room temperature for 15 minutes.

Step 8

8. Pellet

Spin reaction over a 150 ul sucrose cushion (10 mM HEPES, pH 7.7, 50 mM KCl, 20 μM Taxol, 40% sucrose w/v) at 60,000 rpm for 20 minutes at 26°C in an ultracentrifuge.

Step 9

9. Collect Fractions


  • Collect the supernatant and add an equal volume of 2X Sample Buffer to the supernatant fraction.
  • Gently wash the supernatant/cushion interface twice with reaction buffer.
  • Discard the cushion.
  • Gently rinse the pellet twice with reaction buffer.
  • Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.

Step 10

10. Analyze

Analyze fractions by SDS-PAGE.

Protocol


Step 1

1. Assemble

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 @ 20 mg/ml | [final] = 2 mg/ml

Step 2

2. Incubate

Incubate the polymerization mix on ice for 5 minutes. This allows tubulin to bind GTP.

Step 3

3. Clarify

If desired, clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm for 5 minutes at 4°C in a TLA-100 tabletop ultracentrifuge rotor.

Step 4

4. Polymerize

Induce polymerization by incubating the polymerization mix in a 37°C water bath for 1 hour.

Step 5

5. Add Taxol Stepwise

Add Taxol to the polymerized microtubules in the following stepwise manner. Continue incubating in a 37°C water bath:


  • Add 2 µM Taxol at a 1:10 volume ratio (i.e. 5 µl). Incubate for 10 minutes.
  • Add 20 µM Taxol at a 1:10 volume ratio (i.e. 5.5 µl). Incubate for 10 minutes.
  • Add 200 µM Taxol at a 1:10 volume ratio (i.e. 6 µl). 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 tubulin (i.e. 20 µM). Taxol must be included in all subsequent buffers.

Step 6

6. Assemble Reaction

Mix protein of interest with 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).

Step 7

7. Incubate Reaction

Incubate the reaction at room temperature for 15 minutes.

Step 8

8. Pellet

Spin reaction over a 150 ul sucrose cushion (10 mM HEPES, pH 7.7, 50 mM KCl, 20 μM Taxol, 40% sucrose w/v) at 60,000 rpm for 20 minutes at 26°C in an ultracentrifuge.

Step 9

9. Collect Fractions


  • Collect the supernatant and add an equal volume of 2X Sample Buffer to the supernatant fraction.
  • Gently wash the supernatant/cushion interface twice with reaction buffer.
  • Discard the cushion.
  • Gently rinse the pellet twice with reaction buffer.
  • Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.

Step 10

10. Analyze

Analyze fractions by SDS-PAGE.

Overview


At relatively high centripetal forces, polymerized microtubules will pellet while unpolymerized tubulin will remain suspended in solution. This biophysical property can be exploited in probing for microtubule-binding agents and assessing tubulin polymerization competency (when stabilizing factors such as Taxol are excluded).

For general tubulin handling instructions, please see “Storing and Handling Cryopreserved Tubulin” or “Storing and Handling Lyophilized Tubulin.”

Recommended Products


Unlabeled Tubulin


Buffer


Protocol


1. Assemble

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 @ 20 mg/ml | [final] = 2 mg/ml

2. Incubate

Incubate the polymerization mix on ice for 5 minutes. This allows tubulin to bind GTP.

3. Clarify

Clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm for 5 minutes at 4°C in a TLA-100 tabletop ultracentrifuge rotor.

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 2 µM Taxol at a 1:10 volume ratio (i.e. 5 µl). Incubate for 10 minutes.
  • Add 20 µM Taxol at a 1:10 volume ratio (i.e. 5.5 µl). Incubate for 10 minutes.
  • Add 200 µM Taxol at a 1:10 volume ratio (i.e. 6 µl). 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 tubulin (i.e. 20 µM). Taxol must be included in all subsequent buffers.

6. Assemble Reaction

Mix protein of interest with 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).

7. Incubate Reaction

Incubate the reaction at room temperature for 15 minutes.

8. Pellet

Spin reaction over a 150 ul sucrose cushion (10 mM HEPES, pH 7.7, 50 mM KCl, 20 μM Taxol, 40% sucrose w/v) at 60,000 rpm for 20 minutes at 26°C in an ultracentrifuge.

9. Collect Fractions


  • Collect the supernatant and add an equal volume of 2X Sample Buffer to the supernatant fraction.
  • Gently wash the supernatant/cushion interface twice with reaction buffer.
  • Discard the cushion.
  • Gently rinse the pellet twice with reaction buffer.
  • Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.

10. Analyze

Analyze fractions by SDS-PAGE.