文章基本信息

标题：Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide
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作者：Christopher J. Benjamin ; Kyle J. Wright ; Scott C. Bolton 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2016
卷号：6
期号：1
DOI：10.1038/srep32500
语种：English
出版社：Springer Nature
摘要：We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with N(α), N(α)-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.