Purification and Mass-Spectrometry Identification of Microtubule-Binding Proteins from Xenopus Egg Extracts
Microtubule-binding proteins are conveniently divided into two large groups: MAPs (microtubule-associated proteins), which can stabilize, anchor, and/or nucleate microtubules, and motors, which use the energy of ATP hydrolysis for a variety of functions, including microtubule network organization and cargo transportation along microtubules. Here, we describe the use of Taxol-stabilized microtubules for purification of MAPs, motors, and their complexes from Xenopus egg extracts. Isolated proteins are analysed using sodium dodecyl sulfate gel electrophoresis and identified by various mass spectrometry and database mining technologies. Found proteins can be grouped into three classes: (1) known MAPs and motors; (2) proteins previously reported as associated with the microtubule cytoskeleton, but without a clearly defined cytoskeletal function; (3) proteins not yet described as having microtubule localization. Sequence-similarity methods employed for protein identification allow efficient identification of MAPs and motors from species with yet unsequenced genomes.
Key WordsTubulin microtubule microtubule-associated protein MAP motor Xenopus egg extracts mass spectrometry proteomics
- 21.Ausubel, F. M., Brent, R., Kingston, R. E., et al. (2005) Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, NJ.Google Scholar
- 35.Brady, S. T. and Lasek, R. J. (1984) Adenylyl imidodiphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, produces a stable intermediate in the motility cycle of fast axonal transport. Biol. Bull. 167, 503Google Scholar