Abstract
The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid assay based on insights in type I cytokine signal transduction. Bait and prey polypeptides are tethered to mutant cytokine receptor chimeras which are impaired in signaling. On bait-prey interaction and after ligand stimulation, the JAK-STAT signaling cascade is initiated leading to transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In addition to a physiologically relevant context for mammalian protein-protein interactions this method provides separation of interactor and effector zones, and can be applied for both analytical and screening purposes. In the protocol described here, a cytokine receptor derived surface tag is used as a selectable marker. After an initial presort step using magnetic-activated cell sorting (MACS), “positive” cells are selected by fluorescence-activated cell sorting (FACS).
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Lievens, S., Van der Heyden, J., Vertenten, E., Plum, J., Vandekerckhove, J., Tavernier, J. (2004). Design of a Fluorescence-Activated Cell Sorting-Based Mammalian Protein-Protein Interaction Trap. In: Hawley, T.S., Hawley, R.G. (eds) Flow Cytometry Protocols. Methods in Molecular Biology™, vol 263. Humana Press. https://doi.org/10.1385/1-59259-773-4:293
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DOI: https://doi.org/10.1385/1-59259-773-4:293
Publisher Name: Humana Press
Print ISBN: 978-1-58829-234-6
Online ISBN: 978-1-59259-773-4
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