ArticleNature protocols2013
Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.
Article in Nature protocols, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed.
- Mapping the dynamic plant interactome: from in vitro assays to in vivo quantitative approaches.Plant methods · 2026Review
- Analytical techniques for nucleic acid and protein detection with single-molecule sensitivity.Experimental & molecular medicine · 2025Review
- Profiling protein-protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia.Nature biomedical engineering · 2024Article
- Formation of frameshift-stimulating RNA pseudoknots is facilitated by remodeling of their folding intermediates.Nucleic acids research · 2021Article
- Advances in integrative structural biology: Towards understanding protein complexes in their cellular context.Computational and structural biotechnology journal · 2021Review
- Article
- The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab.Biomolecules · 2019Article
- Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET.Proceedings of the National Academy of Sciences of the United States of America · 2017Article
- Coordination among tertiary base pairs results in an efficient frameshift-stimulating RNA pseudoknot.Nucleic acids research · 2017Article
- A quantitative model for the rate-limiting process of UGA alternative assignments to stop and selenocysteine codons.PLoS computational biology · 2017Article
- Single-molecule pull-down for investigating protein-nucleic acid interactions.Methods (San Diego, Calif.) · 2016Article
- PIF1-Interacting Transcription Factors and Their Binding Sequence Elements Determine the in Vivo Targeting Sites of PIF1.The Plant cell · 2016Article
- A starting point for fluorescence-based single-molecule measurements in biomolecular research.Molecules (Basel, Switzerland) · 2014Review
- Utilizing functional cell-free extracts to dissect ribonucleoprotein complex biology at single-molecule resolution.Wiley interdisciplinary reviews. RNAReview
Corrections and comments
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes ∼4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.
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