Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

Cited 54 time in scopus
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Title
Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics
Author(s)
H W Lee; T Kyung; J Yoo; T Kim; C Chung; J Y Ryu; Hanki Lee; K Park; S Lee; W D Jones; D S Lim; C Hyeon; W D Heo; T Y Yoon
Bibliographic Citation
Nature Communications, vol. 4, pp. 1505-1505
Publication Year
2013
Abstract
Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein-protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein-protein interactions as they occur in unpurified cell extracts. By analysing single Ras-Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein-protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein-protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein-protein interaction level.
ISSN
2041-1723
Publisher
Springer-Nature Pub Group
DOI
http://dx.doi.org/10.1038/ncomms2507
Type
Article
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1. Journal Articles > Journal Articles
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