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

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

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Official URL: http://edoc.unibas.ch/dok/A6263208

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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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Stahlberg)
UniBasel Contributors:Stahlberg, Henning
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:American Chemical Society
ISSN:0003-2700
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:20 Jun 2014 07:56
Deposited On:20 Jun 2014 07:56

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