title: Detection of Interaction Between Toll-Like Receptors and Other Transmembrane Proteins by Co-immunoprecipitation Assay.
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.
authors.affiliation: Division of Integrative Biosciences and Biotechnologies, Department of Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.
authors.forename: Yu-Ran
authors.initials: YR
authors.lastname: Lee
authors.affiliation: Division of Integrative Biosciences and Biotechnologies, Department of Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.
authors.forename: Wondae
authors.initials: W
authors.lastname: Kang
authors.affiliation: Division of Integrative Biosciences and Biotechnologies, Department of Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea. youmekim@postech.ac.kr.
authors.forename: You-Me
authors.initials: YM
authors.lastname: Kim
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D051193:Toll-Like Receptors
keywords: Co-immunoprecipitation; Digitonin; TLR9; Toll-like receptors; Transmembrane proteins; UNC93B1