Purification of tubulin and microtubule-associated proteins by membrane ion-exchange chromatography.
Microtubules, composed of tubulin and microtubule-associated proteins (MAPs), can be isolated using routine procedures from homogenates of vertebrate brain. Often, it is necessary then to purify the tubulin from the MAPs, and normally this purification is effected by standard techniques of ion-exchange chromatography. However, such procedures can be expensive, both in the consumption of buffers and other expensive components (e. g. GTP) and in investigator time. Here, we demonstrate that membrane ion exchangers mounted in syringe filter cartridges can be used to separate tubulin from MAPs in a matter of minutes, compared to the several hours that are normally required for typical chromatographic procedures using phosphocellulose orDEAE. The resulting tubulin is competent to assemble into microtubules upon either addition of the purified MAPs or addition of the microtubule-stabilizing drug Taxol. Thus, the procedure should be useful to investigators requiring a rapid and effective purification of tubulin for use in assembly studies or in vitro motility assays.
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, 03755, USA. rds@dartmouth.edu
R D
RD
Sloboda
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LM
Belfi
D000818:Animals; D001923:Brain Chemistry; D002645:Chickens; D002852:Chromatography, Ion Exchange / Q000379:methods*; D008567:Membranes, Artificial; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification*; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D014404:Tubulin / Q000302:isolation & purification*
D008567:Membranes, Artificial; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D014404:Tubulin