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-stabilized microtubule “seeds” can serve as nucleation sites for microtubule elongation. By adding microtubule seeds to polymerization reactions, one can surpass the rate-limiting step of nucleation and specifically assess microtubule elongation. For more information on generating microtubule polymerization mixes visit the Generating GMPCPP-Stabilized Microtubules or Generating Taxol-Stabilized Microtubules protocols.

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-stabilized microtubule “seeds” can serve as nucleation sites for microtubule elongation. By adding microtubule seeds to polymerization reactions, one can surpass the rate-limiting step of nucleation and specifically assess microtubule elongation. For more information on generating microtubule polymerization mixes visit the Generating GMPCPP-Stabilized Microtubules or Generating Taxol-Stabilized Microtubules protocols.

Recommended Products


Unlabeled Tubulin Protein


Labeled Tubulin Protein


Cycled Tubulin Protein Product Image

Cy5 Labeled Tubulin Protein Product Image

Protocol


1. Assemble

Assemble the polymerization mix on ice. Add labeled tubulin if desired. Fluorescent dye-labeled tubulin is typically included at a 1:4 ratio with unlabeled tubulin, although this ratio should be adjusted based on experimental application and specific product labeling stoichiometry ([dye]/[tubulin]). As a benchmark, a final ratio of 1 labeled tubulin heterodimer per 4 unlabeled tubulin heterodimers results in uniform and bright fluorescent microtubules. Biotinylated tubulin is typically included at a 1:50 ratio with unlabeled tubulin.


  • 59 µl H20 | final volume = 100 µl
  • 20 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 1 µl DTT @ 100 mM | [final] = 1 mM
  • 10 µl GMPCPP @ 10 mM | [final] = 1 mM
  • 10 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

Clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100).

Spin at 90k rpm for 5 minutes at 4°C

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-stabilized microtubule seeds are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULE SEEDS ON ICE!

5. Pellet

Pellet the GMPCPP-stabilized microtubule seeds to remove any free GMPCPP and unpolymerized tubulin. Dilute the 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 an ultracentrifuge rotor (i.e. TLA 100).

6. Resuspend

Discard the supernatant and resuspend the pelleted GMPCPP-stabilized microtubule seeds with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to the original volume (i.e. 100 µl). Use a cut pipette tip to avoid shearing the microtubule seeds.

 NOTE :

The GMPCPP-stabilized microtubule seeds are now ready for experimental use and can be kept at room temperature for several days. Microtubule seeds are typically added to subsequent polymerization reactions at a 1:20 ratio to serve as sites of microtubule nucleation.

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-stabilized microtubule “seeds” can serve as nucleation sites for microtubule elongation. By adding microtubule seeds to polymerization reactions, one can surpass the rate-limiting step of nucleation and specifically assess microtubule elongation. For more information on generating microtubule polymerization mixes visit the Generating GMPCPP-Stabilized Microtubules or Generating Taxol-Stabilized Microtubules protocols.

Recommended Products


Unlabeled Tubulin Protein

Unlabeled Tubulin Protein

Cycled Tubulin Protein Product Image

Labeled Tubulin Protein

Cy5 Labeled Tubulin Protein Product Image

Recommended Products


Recommended Products

Unlabeled Tubulin Protein

Cycled Tubulin Protein Product Image

Labeled Tubulin Protein

Cy5 Labeled Tubulin Protein Product Image

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 1:4 ratio with unlabeled tubulin, although this ratio should be adjusted based on experimental application and specific product labeling stoichiometry ([dye]/[tubulin]). As a benchmark, a final ratio of 1 labeled tubulin heterodimer per 4 unlabeled tubulin heterodimers results in uniform and bright fluorescent microtubules. Biotinylated tubulin is typically included at a 1:50 ratio with unlabeled tubulin.


  • 59 µl H20 | final volume = 100 µl
  • 20 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 1 µl DTT @ 100 mM | [final] = 1 mM
  • 10 µl GMPCPP @ 10 mM | [final] = 1 mM
  • 10 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

Clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100).

Spin at 90k rpm for 5 minutes at 4°C
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-stabilized microtubule seeds are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULE SEEDS ON ICE!

Step 5

5. Pellet

Pellet the GMPCPP-stabilized microtubule seeds to remove any free GMPCPP and unpolymerized tubulin. Dilute the 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 an ultracentrifuge rotor (i.e. TLA 100).

Step 6

6. Resuspend

Discard the supernatant and resuspend the pelleted GMPCPP-stabilized microtubule seeds with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to the original volume (i.e. 100 µl). Use a cut pipette tip to avoid shearing the microtubule seeds.

 NOTE :

The GMPCPP-stabilized microtubule seeds are now ready for experimental use and can be kept at room temperature for several days. Microtubule seeds are typically added to subsequent polymerization reactions at a 1:20 ratio 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 1:4 ratio with unlabeled tubulin, although this ratio should be adjusted based on experimental application and specific product labeling stoichiometry ([dye]/[tubulin]). As a benchmark, a final ratio of 1 labeled tubulin heterodimer per 4 unlabeled tubulin heterodimers results in uniform and bright fluorescent microtubules. Biotinylated tubulin is typically included at a 1:50 ratio with unlabeled tubulin.


  • 59 µl H20 | final volume = 100 µl
  • 20 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 1 µl DTT @ 100 mM | [final] = 1 mM
  • 10 µl GMPCPP @ 10 mM | [final] = 1 mM
  • 10 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

Clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100).

Spin at 90k rpm for 5 minutes at 4°C
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-stabilized microtubule seeds are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULE SEEDS ON ICE!

Step 5

5. Pellet

Pellet the GMPCPP-stabilized microtubule seeds to remove any free GMPCPP and unpolymerized tubulin. Dilute the 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 an ultracentrifuge rotor (i.e. TLA 100).

Step 6

6. Resuspend

Discard the supernatant and resuspend the pelleted GMPCPP-stabilized microtubule seeds with room temperature 1X Tubulin PEM Buffer + 1 mM DTT to the original volume (i.e. 100 µl). Use a cut pipette tip to avoid shearing the microtubule seeds.

 NOTE :

The GMPCPP-stabilized microtubule seeds are now ready for experimental use and can be kept at room temperature for several days. Microtubule seeds are typically added to subsequent polymerization reactions at a 1:20 ratio to serve as sites of microtubule nucleation.