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. 2023:2690:193-204.
doi: 10.1007/978-1-0716-3327-4_18.

An Interactome Assay for Detecting Interactions between Extracellular Domains of Receptor Kinases

Jente Stouthamer  1 , Sergio Martin-Ramirez  1 , Elwira Smakowska-Luzan  2
Affiliations

An Interactome Assay for Detecting Interactions between Extracellular Domains of Receptor Kinases

Jente Stouthamer et al. Methods Mol Biol. 2023.

Abstract

Interactions between extracellular domains (ECDs) are crucial for many physiological processes in the cell, most importantly perception of its environment. However, studying these often-transient interactions can be challenging. Here we describe a method that allows for in vitro detection of extracellular domain interactions through an oligomerization-based cell surface interaction (CSI) assay. In a CSI, bait- and prey-tagged proteins are produced and secreted by Drosophila S2 cells to ensure proper folding and post-translational modifications. Subsequently, Bait (FC fragment) and Prey (pentamer domain and alkaline phosphatase) tags allow the detection of interactions in protein A-coated 96 wells plates through a colorimetric readout. Due to the easy detection of interactions this approach can be used for high-throughput screening and mapping of extracellular interaction networks.

Keywords: ECDs; High-throughput screen; Interactome assay; Protein–protein interactions; Receptor kinases; S2 cell protein expression; Signaling.

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