Unknown

Dataset Information

0

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


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.

SUBMITTER: Lee HW 

PROVIDER: S-EPMC3586730 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Lee Hong-Won HW   Kyung Taeyoon T   Yoo Janghyun J   Kim Tackhoon T   Chung Chaeuk C   Ryu Ji Young JY   Lee Hanki H   Park Kihyun K   Lee Sangkyu S   Jones Walton D WD   Lim Dae-Sik DS   Hyeon Changbong C   Heo Won Do WD   Yoon Tae-Young TY  

Nature communications 20130101


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 interacti  ...[more]

Similar Datasets

| S-EPMC8195492 | biostudies-literature
| S-EPMC4421855 | biostudies-other
| S-EPMC7695462 | biostudies-literature
| S-EPMC5293553 | biostudies-literature
| S-EPMC5161575 | biostudies-literature
| S-EPMC5184370 | biostudies-literature
| S-EPMC6507414 | biostudies-literature
| S-EPMC5716724 | biostudies-literature
| S-EPMC1679905 | biostudies-literature
| S-EPMC4022483 | biostudies-literature