Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions
Autor(en): | Beutel, Oliver Roder, Friedrich Birkholz, Oliver Rickert, Christian Steinhoff, Heinz-Juergen Grzybek, Michal Coskun, Uenal Piehler, Jacob |
Stichwörter: | ACTIVATION; BIMOLECULAR FLUORESCENCE COMPLEMENTATION; BINDING-SITE; Chemistry; Chemistry, Multidisciplinary; Chemistry, Physical; DOMAIN; EGF RECEPTOR; fluorescence microscopy; GROWTH-FACTOR RECEPTOR; lipid phase separation; LIPID-BILAYERS; LIVING CELLS; Materials Science; Materials Science, Multidisciplinary; Nanoscience & Nanotechnology; PLASMA-MEMBRANE; polymer-supported membrane; POLYMER-SUPPORTED MEMBRANES; protein-lipid interaction; protein-protein interaction; Science & Technology - Other Topics; signaling complexes | Erscheinungsdatum: | 2015 | Herausgeber: | AMER CHEMICAL SOC | Enthalten in: | ACS NANO | Band: | 9 | Ausgabe: | 10 | Startseite: | 9783 | Seitenende: | 9791 | Zusammenfassung: | We present an ultrasensitive technique for quantitative protein protein interaction analysis in a two-dimensional format based on phase-separated, micropatterned membranes. Interactions between proteins captured to lipid probes via an affinity tag trigger partitioning into the liquid-ordered phase, which is readily quantified by fluorescence imaging. Based on a calibration with well-defined low-affinity protein protein interactions, equilibrium dissociation constants >1 mM were quantified. Direct capturing of proteins from mammalian cell lysates enabled us to detect homo- and heterodimerization of signal transducer and activator of transcription proteins. Using the epidermal growth factor receptor (EGFR) as a model system, quantification of low-affinity interactions between different receptor domains contributing to EGFR dimerization was achieved. By exploitation of specific features of the membrane-based assay, the regulation of EGFR dimerization by lipids was demonstrated. |
ISSN: | 19360851 | DOI: | 10.1021/acsnano.5b02696 |
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geprüft am 06.06.2024