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Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy


Giss, Dominic; Kemmerling, Simon; Dandey, Venkata; Stahlberg, Henning; Braun, Thomas (2014). Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy. Analytical Chemistry, 86(10):4680-4687.

Abstract

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.

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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2 citations in Web of Science®
2 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:Special Collections > SystemsX.ch
Special Collections > SystemsX.ch > Research, Technology and Development Projects > CINA
Special Collections > SystemsX.ch > Research, Technology and Development Projects
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2014
Deposited On:14 Jul 2014 07:08
Last Modified:05 Apr 2016 17:57
Publisher:American Chemical Society
ISSN:0003-2700
Publisher DOI:https://doi.org/10.1021/ac4027803
PubMed ID:24773394
Permanent URL: https://doi.org/10.5167/uzh-97276

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