Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Summary

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

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 Å, 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 spectrometry¹.

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

Protocol

1. Preparing for Crosslinking

1. You will need to plate a sufficient number of cells to allow isolation of 500 μg of protein per standard tube reaction. A single tube may be enough for identification of putative interactors of a protein of interest by immunoblot. In this case bead bound material can be eluted with SDS-PAGE sample buffer (immunoprecipitation). For mass spectrometry analysis, the number of standard reactions should be increased at least ten times and protein complexes should be eluted by out-competition with a peptide antigen recognized by the antibody used (immunoaffinity chromatography). This strategy will allow the isolation of protein com....

Discussion

DSP, a membrane-permeable, chemically reducible crosslinker with a spacer arm of 12 Å is used to stabilize transient protein interactions ^1,2,3,4. Here we exemplified this strategy with the adaptor complex AP-3 a soluble protein complex that recognizes and sorts membrane proteins into vesicles from endosomes ⁵. AP-3 selectively binds to the zinc transporter ZnT3 and the lipid kinase phosphatidylinositol-4-kinase type II alpha but not transferrin receptor ^1,4,6. We expanded these obs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate Buffered Saline	Invitrogen	P4417	Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM
Dithiobis (succinimidyl propionate) (DSP)	Thermo Fisher Scientific, Inc.	22585	Moisture sensitive, store in air tight container 4°C
Dimethyl sulphoxide (DMSO) Hybri-Max	Sigma-Aldrich	D2650
Triton X-100, SigmaUltra	Sigma-Aldrich	T9284
Dynabeads, Sheep anti-Mouse IgG	Invitrogen	110.31	Beads are also available as sheep anti-rabbit
Dyna-Mag-2 magnet	Invitrogen	123-21D