Metallothionein as a clonable tag for protein localization by electron microscopy of cells.
Metallothionein as a clonable tag for protein localization by electron microscopy of cells.
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DOI：
10.1111/jmi.12262
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发表时间：
2015-10
影响因子：
2
通讯作者：
McIntosh JR
中科院分区：
文献类型：
--
作者：
Morphew MK;O'Toole ET;Page CL;Pagratis M;Meehl J;Giddings T;Gardner JM;Ackerson C;Jaspersen SL;Winey M;Hoenger A;McIntosh JR
一个良性的，可克隆的标签定位的蛋白质的电子显微镜细胞将是有价值的，特别是如果它提供高信噪比和良好的空间分辨率的标记。在这里，我们探讨使用金属硫蛋白作为这样的定位标记。我们已经取得了很好的成功，结蛋白在体外标记和酵母纺锤体极体的一个组成部分在细胞中标记。在固定和包埋后或在冷冻置换固定过程中添加的重金属提供了容易可见的信号，而不必担心重原子会影响蛋白质在其生理环境中的行为。然而，我们的方法不适用于核孔复合物的蛋白质组分，这表明这种方法还没有普遍适用。我们提供了我们的最佳标记条件和其他条件的完整描述，希望我们的工作将允许其他人标记他们自己的感兴趣的蛋白质和/或改进我们定义的方法。
A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.