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 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.

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

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 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.

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

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 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.

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

Recommended Products


Unlabeled Tubulin


Labeled Tubulin


Recommended Products


Unlabeled Tubulin


Labeled Tubulin

Recommended Products


Unlabeled Tubulin

Unlabeled Tubulin

Labeled Tubulin

Protocol


Step 1

1. Assemble

Assemble the polymerization mix on ice. Add labeled tubulin if desired. Fluorescent dye-labeled tubulin is typically included at a 1:10 ratio with unlabeled tubulin. Biotinylated tubulin is typically included at a 1:50 ratio with unlabeled tubulin.


  • 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 (optional)

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.

 NOTE :

This is recommended if the tubulin products have undergone an additional freeze-thaw cycle.

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.

At this point, the Taxol-stabilized microtubules can be used directly. However, if it is desirable to remove any unpolymerized tubulin, the Taxol-stabilized microtubules can be pelleted and resuspended in fresh buffer.

 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. Pellet (optional)

Layer the Taxol-stabilized microtubules over a 150 µl pre-warmed glycerol cushion (40% w/v glycerol in 1X Tubulin PEM Buffer) in a TLA 100 tabletop ultracentrifuge rotor. Spin at 90k rpm for 5 minutes at 27°C.

Step 7

7. Resuspend (optional)


  • Discard the supernatant.
  • Gently wash the supernatant/cushion interface twice with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol.
  • Discard the cushion.
  • Gently rinse the pellet twice with prewarmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol.
  • Resuspend the pelleted Taxol-stabilized microtubules with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol to the original volume (i.e. 50 µl).

Protocol


Step 1

1. Assemble

Assemble the polymerization mix on ice. Add labeled tubulin if desired. Fluorescent dye-labeled tubulin is typically included at a 1:10 ratio with unlabeled tubulin. Biotinylated tubulin is typically included at a 1:50 ratio with unlabeled tubulin.


  • 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 (optional)

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.

 NOTE :

This is recommended if the tubulin products have undergone an additional freeze-thaw cycle.

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.

At this point, the Taxol-stabilized microtubules can be used directly. However, if it is desirable to remove any unpolymerized tubulin, the Taxol-stabilized microtubules can be pelleted and resuspended in fresh buffer.

 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. Pellet (optional)

Layer the Taxol-stabilized microtubules over a 150 µl pre-warmed glycerol cushion (40% w/v glycerol in 1X Tubulin PEM Buffer) in a TLA 100 tabletop ultracentrifuge rotor. Spin at 90k rpm for 5 minutes at 27°C.

Step 7

7. Resuspend (optional)


  • Discard the supernatant.
  • Gently wash the supernatant/cushion interface twice with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol.
  • Discard the cushion.
  • Gently rinse the pellet twice with prewarmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol.
  • Resuspend the pelleted Taxol-stabilized microtubules with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol to the original volume (i.e. 50 µl).

Protocol


1. Assemble

Assemble the polymerization mix on ice. Add labeled tubulin if desired. Fluorescent dye-labeled tubulin is typically included at a 1:10 ratio with unlabeled tubulin. Biotinylated tubulin is typically included at a 1:50 ratio with unlabeled tubulin.


  • 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 (optional)

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.

 NOTE :

This is recommended if the tubulin products have undergone an additional freeze-thaw cycle.

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.

At this point, the Taxol-stabilized microtubules can be used directly. However, if it is desirable to remove any unpolymerized tubulin, the Taxol-stabilized microtubules can be pelleted and resuspended in fresh buffer.

 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. Pellet (optional)

Layer the Taxol-stabilized microtubules over a 150 µl pre-warmed glycerol cushion (40% w/v glycerol in 1X Tubulin PEM Buffer) in a TLA 100 tabletop ultracentrifuge rotor. Spin at 90k rpm for 5 minutes at 27°C.

7. Resuspend (optional)


  • Discard the supernatant.
  • Gently wash the supernatant/cushion interface twice with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol.
  • Discard the cushion.
  • Gently rinse the pellet twice with prewarmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol.
  • Resuspend the pelleted Taxol-stabilized microtubules with pre-warmed 1X Tubulin PEM Buffer + 1 mM DTT + 20 µM Taxol to the original volume (i.e. 50 µl).