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research article

Exploring the Interactome: Microfluidic Isolation of Proteins and Interacting Partners for Quantitative Analysis by Electron Microscopy

Giss, Dominic
•
Kemmerling, Simon
•
Dandey, Venkata
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May 20, 2014
Analytical Chemistry

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.

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Type
research article
DOI
10.1021/ac4027803
Author(s)
Giss, Dominic
Kemmerling, Simon
Dandey, Venkata
Stahlberg, Henning  orcid-logo
Braun, Thomas
Date Issued

2014-05-20

Publisher

American Chemical Society (ACS)

Published in
Analytical Chemistry
Volume

86

Issue

10

Start page

4680

End page

4687

Editorial or Peer reviewed

REVIEWED

Written at

OTHER

EPFL units
LBEM  
Available on Infoscience
February 13, 2020
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/165430
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