Separation of human γ-tubulin ring complexes from cell lysates using gel filtration chromatography / 军事医学

ABSTRACT

Objective To separate human γ-tubulin ring complexes (γTuRC) .Methods Cell lysates prepared from 293FT cells were separated using gel filtration chromatography .Then, the eluate fractions containing γTuRC or γ-tubulin small complexes (γTuSC ) were determined by immunoblotting .Results As the constitutive components of γTuRC,γ-tubulin,γ-tubulin complex protein 2 (GCP2), GCP3 and GCP4 were eluted and enriched in the fourth fraction .The molecular mass of eluates in the fourth fraction was about 2000 ×10 3 .Following γTuRC, the constitutive components ofγTuSC including γ-tubulin, GCP2 and GCP3 were eluted and enriched in the fourteenth fraction .The molecular mass of eluates in the fourteenth fraction was about 310 ×10 3 .Unassembled free components were washed out in the eighteenth and subsequent fractions .γTuRC could be detected in the corresponding fractions by negative-PAGE separation .ConclusionγTuRC and γTuSC were successfully separated from the unassembled free components in the fourth ( 4#) and fourteenth (14#) eluted fraction, respectively.The eluates containing ofγTuRC orγTuSC can be used for microtubule assembly research.