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Author Notes:

Correspondence to: Victor Faundez at faundez@cellbio.emory.edu

Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Subjects:

Research Funding:

This work was supported by grants from the National Institutes of Health to V.F. (NS42599 and GM077569).

Keywords:

  • Animals
  • Cells, Cultured
  • Chromatography, Affinity
  • Cross-Linking Reagents
  • Humans
  • Immunomagnetic Separation
  • Protein Interaction Mapping
  • Proteins
  • Succinimides

Isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography.

Tools:

Journal Title:

Journal of Visualized Experiments

Volume:

Volume 2010, Number 37

Publisher:

, Pages 1855-1855

Type of Work:

Article | Final Publisher PDF

Abstract:

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 A, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry.

Copyright information:

© 2010, Journal of Visualized Experiments

This is an Open Access work distributed under the terms of the Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/).

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