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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 protocols Generating GMPCPP Stabilized Microtubules (for short, rigid microtubules) or Generating Taxol Stabilized Microtubules (for long, flexible microtubules).
Protocols
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 protein @ 20 mg/ml | [final] = 2 mg/ml
2. Incubate
Incubate the polymerization mix on ice for 5 minutes. This allows the tubulin protein to bind GMPCPP.
3. Clarify
Clarify the polymerization mix to remove any protein aggregates. Spin at 90k rpm (350,000 x g) for 5 minutes at 4°C in an ultracentrifuge rotor (i.e. TLA 100). Recover the supernatant on ice in a clean microtube.
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!
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 (350,000 x g) 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.
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.
