Fig. 8.

MT preparation. (a) Addition of GTP to free tubulin (αβ-tubulin dimers) induces MT polymerization (promoted by addition of glycerol [40, 41] and 37°C temperature). When preparing fluorescent MTs, the small amount <5 %) of tubulin labeled with the organic fluorophore Cy3 incorporates randomly into the MT lattice and is distributed sparsely enough that the dye molecules do not affect motor interaction with the MTs in the optical trapping assay. Initially, MTs exhibit dynamic instability [42, 43]. Paclitaxel greatly enhances polymerization and stabilizes the MTs [44-49] (the DMSO in which paclitaxel is dissolved also enhances MT polymerization [50]). (b) Removal of residual-free tubulin and very short MT fragments is accomplished by sedimentation through a 60 %-glycerol “cushion.” First, MTs are layered carefully on top of the cushion. Following centrifugation, the free tubulin and very short MTs remaining in the supernatant are removed. The MT pellet is then resuspended in buffer. These MTs are stable for days to weeks at room temperature