Immunoelectron Microscopy of Cryofixed Freeze-Substituted Saccharomyces cerevisiae
Immunoelectron Microscopy of Cryofixed Freeze-Substituted Saccharomyces cerevisiae
复制标题
DOI：
10.1007/978-1-60761-783-9_15
复制
发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Goldberg, Martin W.
中科院分区：
文献类型：
--
作者：
Fiserova, Jindriska;Goldberg, Martin W.
免疫标记电子显微镜是一项具有挑战性的技术，需要完美的超微结构和抗原保存。高压冷冻是固定细胞结构的理想方法。然而，由于蛋白质的抗原性或可及性丧失，标记的频率很低，因此它在免疫标记中的应用仍然有限。在这里，我们提出了一种免疫金标记法，在保持最好的结构细节的同时，给出了特定的和多重的标记酿酒酵母。我们使用该方案来揭示单个核孔复合体蛋白的组织和与实际运输事件有关的运输因子在酵母中的位置。
Immunolabelling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an ideal way to fix cellular structure. However, its use for immunolabelling has remained limited because of the low frequency of labelling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labelling of the yeast Saccharomyces cerevisiae that gives specific and multiple labelling while keeping the finest structural details. We use the protocol to reveal the organisation of individual nuclear pore complex proteins and the position of transport factors in the yeast S. cerevisiae in relation to actual transport events.