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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 protein concentration can be altered to influence the number and length of microtubules upon polymerization. In general, a tubulin protein concentration of 2 mg/ml results in many short microtubules, while a tubulin protein concentration of 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. To generate long, flexible microtubules, see Generating Taxol-Stabilized 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 1 hour.
NOTE :
After polymerizing, GMPCPP-stabilized microtubules are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULES ON ICE!
After polymerizing, GMPCPP-stabilized microtubules are to be handled at room temperature. DO NOT PLACE POLYMERIZED MICROTUBULES ON ICE!
5. Pellet
Pellet the GMPCPP-stabilized microtubules to remove any free GMPCPP and unpolymerized tubulin protein. Dilute the microtubules 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 microtubules 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 microtubules.
NOTE :
The GMPCPP-stabilized microtubules are now ready for experimental use and can be kept at room temperature for several days. Microscopy applications often require further diluting the microtubules in 1X Tubulin PEM Buffer + 1 mM DTT until the desired density is achieved.
The GMPCPP-stabilized microtubules are now ready for experimental use and can be kept at room temperature for several days. Microscopy applications often require further diluting the microtubules in 1X Tubulin PEM Buffer + 1 mM DTT until the desired density is achieved.
