Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer.
Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.
Castoldi
Mirco
M
European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Popov
Andrei V
AV
D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001923:Brain Chemistry; D002021:Buffers; D002482:Cellulose / Q000031:analogs & derivatives*; D002853:Chromatography, Liquid; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D001704:Biopolymers; D002021:Buffers; D014404:Tubulin; D002482:Cellulose; C008881:phosphocellulose