Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence.

Abstract

Short microtubule seeds are constructed using heavily rhodamine-labeled tubulin. Polymerisation off the ends of these seeds is initiated using a mixture of 1:10 labeled and unlabeled tubulin, so that the new polymerisation is only dimly labeled. This is done in the presence of NEM tubulin, which inhibits growth from the microtubule minus ends. The polarity-marked microtubules are fixed at a desired length by adding taxol.