Total Internal Reflection Fluorescence (TIRF) Single-Molecule Assay to Analyze the Motility of Kinesin

The motile parameters of kinesin superfamily proteins are fundamental to intracellular transport. Single-molecule motility assays using total internal reflection fluorescence (TIRF) microscopy are a gold standard technique for measuring the motile parameters of kinesin motors. With this technique, one can evaluate the velocity, run length, and binding frequency of kinesins on microtubules by directly observing their motility. This protocol provides a comprehensive procedure for single molecule assays of kinesins, including the preparation of labeled microtubules, the measurement of kinesin motility via TIRF microscopy, and the quantification of kinesin motor parameters. Key features • Analysis of the motility of kinesin superfamily proteins using TIRF microscopy. • In vitro reconstitution using purified microtubules and motors. • Direct measurement of motile parameters of kinesins.