Generating GMPCPP-Stabilized Microtubules

tubulin protein polymerization mix

Tubulin polymerization mixes containing Alexa 488-labeled tubulin (left), Alexa 594-labeled tubulin (center), and Cy5-labeled tubulin (right) at a 1:4 ratio with unlabeled tubulin.

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.

Protocol