Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide

被引：27

作者：

Benjamin, Christopher J. ^{[1
]}

Wright, Kyle J. ^{[1
]}

Bolton, Scott C. ^{[1
]}

Hyun, Seok-Hee ^{[1
]}

Krynski, Kyle ^{[1
]}

Grover, Mahima ^{[1
]}

Yu, Guimei ^{[2
]}

Guo, Fei ^{[2
]}

Kinzer-Ursem, Tamara L. ^{[3
]}

Jiang, Wen ^{[2
]}

Thompson, David H. ^{[1
,3
,4
]}

机构：

[1] Purdue Univ, Dept Chem, W Lafayette, IN 47907 USA

[2] Purdue Univ, Dept Biol Sci, W Lafayette, IN 47907 USA

[3] Purdue Univ, Weldon Sch Biomed Engn, W Lafayette, IN 47907 USA

[4] Purdue Univ, Ctr Canc Res, W Lafayette, IN 47907 USA

来源：

SCIENTIFIC REPORTS | 2016年 / 6卷

关键词：

CRYO-EM STRUCTURE; ANGSTROM RESOLUTION; SINGLE; GROEL; COMPLEXES; SHEETS; LIPIDS;

D O I：

10.1038/srep32500

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with N-alpha, N-alpha-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 angstrom. 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.

引用

页数：11

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