Grid coatings for capture of proteins and other compounds

Patent metadata

Field	Value
Publication number	US-10989681-B2
Application number	US-201616063029-A
Country	US
Kind code	B2
Filing date	Dec 16, 2016
Priority date	Dec 17, 2015
Publication date	Apr 27, 2021
Grant date	Apr 27, 2021

Abstract

Grids comprising a coating modified with one or more capture agents and a deactivating agent are disclosed. Methods of using such grids in connection with suitable microscopy techniques, such as for determining the structure of target compounds including proteins, are also disclosed.

First claim

The invention claimed is:

1. A grid for elucidating high-resolution structure of target proteins comprising a coating modified with a capture agent and further comprising a deactivating agent, wherein the coating comprises a graphene oxide sheet modified with Nα, Nα-dicarboxymethyllysine (GO-NTA), wherein the capture agent is Nα, Nα-dicarboxymethyllysine and the deactivating agent is methoxy-poly(ethylene glycol 350)-1,2-distearoyl-sn-3-glycerophosphoethanolamine.

2. The grid of claim 1 , wherein the coating is a monolayer.

3. The grid of claim 1 , wherein the coating comprises a plurality of micro-porous or nano-porous graphene oxide sheets.

4. The grid of claim 3 , further comprising one or more lipid monolayers wherein the monolayers are deposited onto the graphenic layer residing on the grid.

5. The grid of claim 1 , wherein the coating is between about 0.1 nanometers and 100 microns thick.

6. The grid of claim 5 , wherein the coating is between about 0.1 nanometers and 100 nanometers thick.

7. The grid of claim 6 , wherein the coating is between about 0.1 nanometers and 10 nanometers thick.

8. The grid of claim 7 wherein the coating is between about 0.1 nanometers and 2 nanometers thick.

9. The grid of claim 8 , wherein the coating is between about 0.1 nanometers and 1 nanometer thick.

10. The grid of claim 1 , wherein the modification to the coating is done by chemical synthesis.

11. The grid of claim 1 , wherein the target proteins are tagged with polyhistidine.

12. The grid of claim 1 , wherein the target proteins are proteins obtained from a cell lysate.

13. The grid of claim 12 , wherein the cell lysate has been clarified.

14. The grid of claim 1 , wherein said grid is used as a substrate for single particle protein structure analysis by a suitable transmission electron microscope at a resolution of less than or equal to 20 Angstroms.

15. The grid of claim 14 , wherein the resolution is between 1 Angstroms and 10 Angstroms.

16. The grid of claim 15 , wherein the resolution is between 8 Angstroms and 9 Angstroms.

17. A method of preparing target proteins for structure elucidation comprising contacting a grid of claim 1 with a cell lysate comprising target proteins and subjecting the target proteins to a suitable scanning or transmission electron microscopy for single particle structure analysis at a resolution of between 1 and 10 Angstroms.

Assignees

• Purdue Research Foundation

Inventors

• Thompson David H
• Benjamin Christopher
• Wright Kyle J
• Bolton Scott C
• Hyun Seok-Hee

Classifications

• H01J37/20 Primary

  Means for supporting or positioning the object or the material; Means for adjusting diaphragms or lenses associated with the support · CPC title

• B82Y40/00

  Manufacture or treatment of nanostructures · CPC title

• G01N2223/418

  electron microscope · CPC title

• G01N27/4145

  specially adapted for biomolecules, e.g. gate electrode with immobilised receptors · CPC title

• B82Y30/00

  Nanotechnology for materials or surface science, e.g. nanocomposites · CPC title