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

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 an...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2014-05, Vol.86 (10), p.4680-4687
Main Authors: Giss, Dominic, Kemmerling, Simon, Dandey, Venkata, Stahlberg, Henning, Braun, Thomas
Format: Article
Language:English
Subjects:
Animals
Antibodies
Beads
Biochemistry
Biotinylation
Cell Line
Cellular
Cellular biology
Cricetinae
Dynamic structural analysis
Electron microscopy
Humans
Magnetics
Microfluidics
Microfluidics - methods
Microscopy, Electron, Transmission
Molecules
Physiology
Protein Interaction Mapping - methods
Proteins
Proteins - isolation & purification
Quantitative analysis
Transmission electron microscopy
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac4027803