Conjugation of fluorophores to tubulin
Conjugation of fluorophores to tubulin
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DOI：
10.1038/nmeth0405-299
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发表时间：
2005-04-01
期刊：
影响因子：
48
通讯作者：
Borisy, G
中科院分区：
文献类型：
--
作者：
Peloquin, J;Komarova, Y;Borisy, G
使用荧光标记蛋白作为活细胞结构和功能的探针始于对细胞骨架成分的研究(1-4)，并已成为研究这些成分的分布和动态的有力方法(5-7)。细胞骨架的主要成分如微管蛋白、肌动蛋白和肌球蛋白含量丰富，并且可以相对容易地从动物组织中纯化出数百毫克的量(8-11)。含有胺或硫醇反应基团的荧光团分别用于选择性修饰赖氨酸或半胱氨酸残基。重要的是蛋白质的衍生化不会导致功能丧失。衍生化后通常使用选择活性蛋白和去除未结合荧光团的方法。在任何给定的荧光发射波长下选择荧光团应优化荧光强度和光稳定性。微管蛋白以聚合形式标记，以保护对组装很重要的残基，并且可以成功地与胺反应性琥珀酰亚胺酯的荧光衍生物缀合。功能性微管蛋白通过聚合和解聚循环来选择。通过修改反应物的比例，Cy3 标记方案还可用于使用 Cy3.5、Cy5、四甲基罗丹明、X-罗丹明、Oregon Green 488 以及 Alexa 488、568 和 594 琥珀酰亚胺酯衍生化微管蛋白。尽管荧光融合蛋白已广泛用于成像研究，但荧光团标记的蛋白仍然是研究细胞骨架动力学和功能的最通用和最直接的工具。该协议描述了从脑组织中分离出的微管蛋白的荧光缀合。
The use of fluorescently labeled proteins as probes of the structure and function of living cells began with the study of the components of the cytoskeleton(1-4) and has become a powerful approach for studying the distribution and dynamics of these components(5-7). Primary components of the cytoskeleton such as tubulin, actin and myosin are abundant and can be purified relatively easily in hundreds of milligram quantities from animal tissues(8-11). Fluorophores containing amine or thiol reactive groups are used to selectively modify Lysine or cysteine residues, respectively. It is essential that derivatization of the protein not result in toss of function. Methods for selecting active protein and removing unconjugated fluorophore are usually used after derivatization. The choice of fluorophore at any given wavelength of fluorescence emission should optimize fluorescence intensity and photostability. Tubulin is Labeled in polymeric form to protect residues that are important for assembly and can be successfully conjugated with fluorescent derivatives of amine-reactive succinimidyl esters. Functional tubulin is selected by cycles of polymerization and depolymerization. By modifying the ratio of reactants, the Cy3-labeling protocol can also be used to derivatize tubulin with Cy3.5, Cy5, tetramethylrhodamine, X-rhodamine, Oregon Green 488, and Alexa 488, 568 and 594 succinimidyl esters. Although fluorescent fusion proteins have become widely used in imaging studies, fluorophore-labeled proteins remain the most versatile and direct toots for studying the dynamics and function of the cytoskeleton. This protocol describes the fluorescent conjugation of tubulin isolated from brain tissue.