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

• Type of Material: Article
• Authors: Stephanie Zlatic, Emory University; Pearl V. Ryder, Emory University; Gloria Salazar, Emory University; Victor Faundez, Emory University
• Language: English
• Date: 2010-12-01
• Publisher: Journal of Visualized Experiments (JoVE)
• Publication Version: Final Published Version
• Copyright Statement: © 2010, Journal of Visualized Experiments
• License: Creative Commons Attribution 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.3791/1855
• Title of Journal or Parent Work: Journal of Visualized Experiments
• ISSN: 1940-087X
• Volume: 2010
• Issue: 37
• Start Page: 1855
• End Page: 1855
• Grant/Funding Information: This work was supported by grants from the National Institutes of Health to V.F. (NS42599 and GM077569).
• Keywords: Proteins; Cross-Linking Reagents; Chromatography, Affinity; Animals; Cells, Cultured; Immunomagnetic Separation; Protein Interaction Mapping; Humans; Succinimides
• Research Categories: Biology, Cell; Health Sciences, General

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.