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

نویسندگان

  • Christopher J. Benjamin
  • Kyle J. Wright
  • Scott C. Bolton
  • Seok-Hee Hyun
  • Kyle Krynski
  • Mahima Grover
  • Guimei Yu
  • Fei Guo
  • Tamara L. Kinzer-Ursem
  • Wen Jiang
  • David H. Thompson
چکیده

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

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عنوان ژورنال:

دوره 6  شماره 

صفحات  -

تاریخ انتشار 2016