Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions

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• 作者：Oliver Beutel ; Friedrich Roder ; Oliver Birkholz ; Christian Rickert ; Heinz-J眉rgen Steinhoff ; Micha艂 Grzybek ; 脺nal Coskun ; Jacob Piehler
• 刊名：ACS Nano
• 出版年：2015
• 出版时间：October 27, 2015
• 年：2015
• 卷：9
• 期：10
• 页码：9783-9791
• 全文大小：588K
• ISSN：1936-086X

文摘

We present an ultrasensitive technique for quantitative protein鈥損rotein 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鈥損rotein 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.