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

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

SUBMITTER: Benjamin CJ 

PROVIDER: S-EPMC5066248 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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

Benjamin Christopher J CJ   Wright Kyle J KJ   Bolton Scott C SC   Hyun Seok-Hee SH   Krynski Kyle K   Grover Mahima M   Yu Guimei G   Guo Fei F   Kinzer-Ursem Tamara L TL   Jiang Wen W   Thompson David H DH  

Scientific reports 20161017

We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with N<sup>α</sup>, N<sup>α</sup>-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 His<sub>6</sub>-T7 bacteriophage and His<sub>6</sub>-GroEL directly from cell lysates. To demonstrat  ...[more]

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