Fig. 7.

Labeling of tubulin with Cy3 dye. Tubulin is first polymerized into MTs. Cy3 conjugated to the reactive N-hydroxysuccinimide (NHS) is then added, which facilitates attachment of the dye to amine groups on the surface of the MT. After pelleting the MTs and removing excess dye, the MTs are resuspended and depolymerized in the cold. The insoluble fraction is then pelleted and the supernatant transferred to a new tube in which the tubulin is repolymerized, pelleted, and depolymerized again, followed by aliquotting and snap freezing. This strategy ensures that the tubulin is labeled in regions other than the key interfaces required for polymerization and that the final product contains only tubulin capable of cyclic polymerization and depolymerization (and thus unperturbed by the attached dye)