NON-FOULING AFFINITY PLATFORMS FOR PROTEIN IMMOBILIZATION IN ELECTRON MICROSCOPY

Single-particle reconstruction has grown significantly with the improvements in various data collection and computational strategies including CTF fitting, the use of vitrified samples and the utilization of ultra-sensitive direct electron detectors. Although these improvements have contributed significantly to the recent evolution of 3D reconstruction analysis, the way samples are prepared for electron microscopy has remained largely unchanged. We report the development of TEM grids that are modified with non-fouling coatings bearing surface grafted nitrilotriacetic acid substituents that promotes specific capture of protein targets for high resolution TEM analysis. The utilization of these grids for specific adsorption of the targeted protein onto the grid surface results in well-controlled surface concentration enhancements and a days-to-minutes reduction in time required for the preparation of a purified sample for cryoEM analysis from an E. coli expression system. The selective and reversible capture of his-tag T7 bacteriophage, RplL, and GroEL from crude lysates, as well as purified nanodisc-solubilized his-malFGK2, on these NTA-modified grids with an exceptionally low level of adsorption by non-target proteins has been observed. Our data illustrates the utility of these grids for selective capture from complex mixtures, detergent-solubilized membrane protein isolates, and expression systems yielding low copy numbers of the desired target in a manner that is well-suited for single particle reconstruction analysis.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Year

2015

Author dc:creator

• Benjamin, Christopher Jerome

Contributors dc:contributor

• David H Thompson
• Kavita Shah
• Jean Chmielewski
• Chittaranjan Das

• Surface Chemistry

Repository record dc:identifier

https://docs.lib.purdue.edu/open_access_dissertations/1091

OAI identifier oai:identifier

oai:docs.lib.purdue.edu:open_access_dissertations-2307