Detection of interaction between toll-like receptors and other transmembrane proteins by co-immunoprecipitation assay

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Toll-like receptors are type I membrane proteins and bind other membrane proteins often via a specific interaction between trans membrane 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.
Publisher
Humana Press Inc.humana@humanapr.com
Issue Date
2016
Language
English
Article Type
Book Chapter
Citation

Methods in Molecular Biology, v.1390, pp.107 - 120

ISSN
1064-3745
DOI
10.1007/978-1-4939-3335-8_7
URI
http://hdl.handle.net/10203/280766
Appears in Collection
MSE-Journal Papers(저널논문)
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