Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.

Details

Serval ID
serval:BIB_77C82DE8C722
Type
Article: article from journal or magazin.
Collection
Publications
Title
Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.
Journal
Proceedings of the National Academy of Sciences of the United States of America
Author(s)
Schmidli C., Albiez S., Rima L., Righetto R., Mohammed I., Oliva P., Kovacik L., Stahlberg H., Braun T.
ISSN
1091-6490 (Electronic)
ISSN-L
0027-8424
Publication state
Published
Issued date
23/07/2019
Peer-reviewed
Oui
Volume
116
Number
30
Pages
15007-15012
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.
Keywords
Biotinylation, Cryoelectron Microscopy/instrumentation, Cryoelectron Microscopy/methods, HeLa Cells, Humans, Image Processing, Computer-Assisted/statistics & numerical data, Imaging, Three-Dimensional, Immunoglobulin Fab Fragments/chemistry, Microfluidic Analytical Techniques/methods, Proteasome Endopeptidase Complex/chemistry, Proteasome Endopeptidase Complex/isolation & purification, Proteasome Endopeptidase Complex/ultrastructure, Protein Conformation, Protein Subunits/chemistry, Protein Subunits/isolation & purification, Vitrification, cryo-EM, endogenous, microfluidics, protein purification, sample preparation
Pubmed
Web of science
Open Access
Yes
Create date
09/06/2023 16:02
Last modification date
08/07/2023 6:50
Usage data