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Detection of Interaction Between Toll-Like Receptors and Other Transmembrane Proteins by Co-immunoprecipitation Assay

  • Yu-Ran Lee
  • Wondae Kang
  • You-Me KimEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1390)

Abstract

Toll-like receptors are type I membrane proteins and bind other membrane proteins often via a specific interaction between transmembrane domains. The co-immunoprecipitation assay is a widely used biochemical technique for assessing interactions among proteins in cell lysates or tissue extracts. By isolating a native protein complex with a specific antibody against a protein of interest, followed by western blotting with an antibody for a binding partner, the co-immunoprecipitation assay can be used to confirm a putative interaction between two proteins. The co-immunoprecipitation assay can also be combined with a proteomics approach such as protein mass spectrometry to build an interactome of a target protein. Despite its usefulness and popularity to probe protein interactions within complex biological samples, the co-immunoprecipitation assay of membrane proteins is rather tricky, often resulting in false data. Here, we describe a co-immunoprecipitation method for analyzing interactions between toll-like receptors and other membrane proteins, using the interaction between TLR9 and UNC93B1 as an example. Especially, we describe an optimal cell lysis and sample preparation method to preserve protein interactions mediated by transmembrane domains.

Key words

Co-immunoprecipitation Transmembrane proteins Toll-like receptors TLR9 UNC93B1 Digitonin 

Notes

Acknowledgement

This work was supported by the grant from National Research Foundation of Korea (NRF-2013R1A1A2074573) and BK21 Plus (10Z20130012243).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Integrative Biosciences and Biotechnologies, Department of Life SciencesPohang University of Science and TechnologyPohangRepublic of Korea

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