Generating GMPCPP Microtubule Seeds

Overview


GMPCPP is a slowly hydrolyzing analog of GTP, and is therefore used to generate stable microtubules that resist depolymerization at room temperature. Short GMPCPP microtubule “seeds” can serve as nucleation sites for microtubule elongation. Therefore, one can utilize microtubule GMPCPP seeds to surpass the rate-limiting step of nucleation and specifically assess microtubule elongation. In this scenario GMPCPP microtubule seeds are typically added to polymerization reactions at a 1:20 volume ratio.

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. Short GMPCPP microtubule “seeds” can serve as nucleation sites for microtubule elongation. Therefore, one can utilize microtubule GMPCPP seeds to surpass the rate-limiting step of nucleation and specifically assess microtubule elongation. In this scenario GMPCPP microtubule seeds are typically added to polymerization reactions at a 1:20 volume ratio.

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

Recommended Products


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Unlabeled Tubulin


Labeled Tubulin


Recommended Products

Unlabeled Tubulin

Labeled Tubulin

Recommended Products


Unlabeled Tubulin

Unlabeled Tubulin

Labeled Tubulin

Recommended Products

Unlabeled Tubulin

CYCLED TUBULIN
( Cat.No. 032005 )
Labeled Tubulin

ALEXA FLUOR® 488 ( Cat.No. 048805 )

ALEXA FLUOR® 594 ( Cat.No. 059405 )

ALEXA FLUOR® 647 ( Cat.No. 064705 )

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.


  • 34 µl H20 | final volume = 50 µl
  • 10 µl Tubulin PEMBuffer @ 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 20 minutes. The incubation time can be varied to influence the length of the microtubule seeds.

 NOTE :

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

5. Pellet

Pellet the GMCPP microtubule seeds to remove any free GMPCPP and unpolymerized tubulin. Dilute the GMPCPP microtubule seeds 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

Discard the supernatant and resuspend the pelleted GMPCPP microtubule seeds with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to the original volume (i.e. 50 µl). At this point the GMPCPP microtubule seeds can be added to subsequent polymerization reactions to serve as sites of microtubule nucleation.

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.


  • 34 µl H20 | final volume = 50 µl
  • 10 µl Tubulin PEMBuffer @ 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 20 minutes. The incubation time can be varied to influence the length of the microtubule seeds.

 NOTE :

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

Step 5

5. Pellet

Pellet the GMCPP microtubule seeds to remove any free GMPCPP and unpolymerized tubulin. Dilute the GMPCPP microtubule seeds 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

Discard the supernatant and resuspend the pelleted GMPCPP microtubule seeds with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to the original volume (i.e. 50 µl). At this point the GMPCPP microtubule seeds can be added to subsequent polymerization reactions to serve as sites of microtubule nucleation.

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.


  • 34 µl H20 | final volume = 50 µl
  • 10 µl Tubulin PEMBuffer @ 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 20 minutes. The incubation time can be varied to influence the length of the microtubule seeds.

 NOTE :

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

Step 5

5. Pellet

Pellet the GMCPP microtubule seeds to remove any free GMPCPP and unpolymerized tubulin. Dilute the GMPCPP microtubule seeds 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

Discard the supernatant and resuspend the pelleted GMPCPP microtubule seeds with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to the original volume (i.e. 50 µl). At this point the GMPCPP microtubule seeds can be added to subsequent polymerization reactions to serve as sites of microtubule nucleation.