Sloboda R D, Belfi L M
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, 03755, USA.
Protein Expr Purif. 1998 Jul;13(2):205-9. doi: 10.1006/prep.1998.0902.
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.
微管由微管蛋白和微管相关蛋白（MAPs）组成，可通过常规程序从脊椎动物脑匀浆中分离出来。通常，之后需要从MAPs中纯化微管蛋白，而这种纯化通常通过离子交换色谱标准技术来实现。然而，此类程序在缓冲液和其他昂贵成分（如GTP）的消耗以及研究人员的时间方面都可能很昂贵。在这里，我们证明安装在注射器滤筒中的膜离子交换器可在几分钟内将微管蛋白与MAPs分离，而使用磷酸纤维素或DEAE的典型色谱程序通常需要数小时。所得的微管蛋白在添加纯化的MAPs或添加微管稳定药物紫杉醇后都能够组装成微管。因此，该程序对于需要快速有效纯化微管蛋白以用于组装研究或体外运动分析的研究人员应该是有用的。
