Isolation and Analysis of Microtubules and Associated Proteins

Abstract

Microtubules, microtubule-associated proteins (MAPs), and motor proteins are essential components of all eukaryotic cells. They are all involved in mitosis and in the movement of organelles, proteins, and vesicles in cells. MAPs act as structural elements of the microtubule component of the cytoskeleton, whereas molecular motors propel cargo along microtubule tracks or translocate microtubules in the cytoplasm. This introduction provides an overview of procedures developed by many labs to isolate microtubules from cell homogenates, purify tubulin, MAPs, and motor proteins from microtubules preparations, and analyze kinesin and cytoplasmic dynein activity by video-enhanced differential interference contrast microscopy and fluorescence microscopy. These ingenious microscope-based assays, which were developed to determine the motility characteristics of kinesin and dynein, reveal, in clear and dramatic fashion, the activity of these amazing nanomachines in real time.