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


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 1:4 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).

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

Unlabeled Tubulin

Labeled Tubulin

Recommended Products


Recommended Products

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 1:4 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).

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).

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.