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
Institution
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
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
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