Generating GMPCPP-Stabilized Microtubules

Overview


GMPCPP is a slowly hydrolyzing analog of GTP, and is therefore used to generate stable microtubules that resist depolymerization at room temperature. The tubulin concentration can be altered to influence the number and length of microtubules upon polymerization. In general, tubulin at 2 mg/ml results in many short microtubules, while tubulin at 0.2 mg/ml results in few long microtubules. In the latter scenario, GMPCPP microtubule seeds are often added to overcome the kinetic barrier of microtubule nucleation. See “Generating GMPCPP Microtubule Seeds” for details.

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

Overview


GMPCPP is a slowly hydrolyzing analog of GTP, and is therefore used to generate stable microtubules that resist depolymerization at room temperature. The tubulin concentration can be altered to influence the number and length of microtubules upon polymerization. In general, tubulin at 2 mg/ml results in many short microtubules, while tubulin at 0.2 mg/ml results in few long microtubules. In the latter scenario, GMPCPP microtubule seeds are often added to overcome the kinetic barrier of microtubule nucleation. See “Generating GMPCPP Microtubule Seeds” for details.

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

Overview


GMPCPP is a slowly hydrolyzing analog of GTP, and is therefore used to generate stable microtubules that resist depolymerization at room temperature. The tubulin concentration can be altered to influence the number and length of microtubules upon polymerization. In general, tubulin at 2 mg/ml results in many short microtubules, while tubulin at 0.2 mg/ml results in few long microtubules. In the latter scenario, GMPCPP microtubule seeds are often added to overcome the kinetic barrier of microtubule nucleation. See “Generating GMPCPP Microtubule Seeds” for details.

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

Recommended Products


Unlabeled Tubulin


Labeled Tubulin


Recommended Products


Unlabeled Tubulin

Unlabeled Tubulin

Labeled Tubulin

Recommended Products


Recommended Products

Unlabeled Tubulin

Labeled Tubulin

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 GMPCPP @ 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 GMPCPP.

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.

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

 NOTE :

After polymerizing, GMPCPP microtubules are to be handled at room temperature.

5. Pellet (optional)

Dilute the GMPCPP microtubules with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to a total volume of 200 µl. Spin at 90k rpm for 5 minutes at 27°C in a TLA-100 tabletop ultracentrifuge rotor.

6. Resuspend (optional)

Discard the supernatant and resuspend the pelleted GMPCPP microtubules with room temperature 1X Tubulin PEM Buffer + 1 mM DTT 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 GMPCPP @ 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 GMPCPP.

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.

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

 NOTE :

After polymerizing, GMPCPP microtubules are to be handled at room temperature.

Step 5

5. Pellet (optional)

Dilute the GMPCPP microtubules with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to a total volume of 200 µl. Spin at 90k rpm for 5 minutes at 27°C in a TLA-100 tabletop ultracentrifuge rotor.

Step 6

6. Resuspend (optional)

Discard the supernatant and resuspend the pelleted GMPCPP microtubules with room temperature 1X Tubulin PEM Buffer + 1 mM DTT 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 GMPCPP @ 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 GMPCPP.

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.

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

 NOTE :

After polymerizing, GMPCPP microtubules are to be handled at room temperature.

Step 5

5. Pellet (optional)

Dilute the GMPCPP microtubules with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to a total volume of 200 µl. Spin at 90k rpm for 5 minutes at 27°C in a TLA-100 tabletop ultracentrifuge rotor.

Step 6

6. Resuspend (optional)

Discard the supernatant and resuspend the pelleted GMPCPP microtubules with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to the original volume (i.e. 50 µl).