共 49 条
Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions
被引:7
作者:
Beutel, Oliver
[1
]
Roder, Friedrich
[1
]
Birkholz, Oliver
[1
]
Rickert, Christian
[2
]
Steinhoff, Heinz-Juergen
[2
]
Grzybek, Michal
[3
,4
]
Coskun, Uenal
[3
,4
]
Piehler, Jacob
[1
]
机构:
[1] Univ Osnabruck, Dept Biol, D-49074 Osnabruck, Germany
[2] Univ Osnabruck, Dept Phys, D-49076 Osnabruck, Germany
[3] Tech Univ Dresden, Univ Clin Carl Gustav Carus, Helmholtz Ctr Munich, Paul Langerhans Inst Dresden, D-01307 Dresden, Germany
[4] German Ctr Diabet Res DZD, D-85764 Neuherberg, Germany
来源:
关键词:
protein-protein interaction;
polymer-supported membrane;
lipid phase separation;
fluorescence microscopy;
signaling complexes;
protein-lipid interaction;
BIMOLECULAR FLUORESCENCE COMPLEMENTATION;
POLYMER-SUPPORTED MEMBRANES;
GROWTH-FACTOR RECEPTOR;
LIVING CELLS;
EGF RECEPTOR;
PLASMA-MEMBRANE;
LIPID-BILAYERS;
BINDING-SITE;
DOMAIN;
ACTIVATION;
D O I:
10.1021/acsnano.5b02696
中图分类号:
O6 [化学];
学科分类号:
0703 ;
摘要:
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.
引用
收藏
页码:9783 / 9791
页数:9
相关论文