Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions

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.

Single‐Molecule Co‐Immunoprecipitation Reveals Functional Inheritance of EGFRs in Extracellular Vesicles

“…In this report, we asked how distinct types of EVs represent their parental cells in terms of their EGFR signaling capacity. Specifically, using our single‐molecule co‐immunoprecipitation (co‐IP) approach, we confirmed meaningful amounts of EGFRs in both the types of EVs released from lung adenocarcinoma cell lines. We characterized the protein–protein interactions (PPIs) of these EGFRs carried by two types of EVs with representative downstream effector proteins.…”

“…Single‐Molecule Co‐IP : Detailed protocols for single molecule co‐IP and imaging are described in refs. . Briefly, quartz slides and coverslips were cleansed and passivated.…”

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

“…However, single-molecule spectroscopy can be a very powerful tool because it can probe subpopulations in a mixture without the need for separation. Single-molecule spectroscopy has been successfully used for characterizing individual molecular states such as the folded and unfolded states of proteins (7)(8)(9), intermediate states (10,11), and transition paths (12)(13)(14)(15)(16)(17) and for identifying specific molecular species and complexes (18)(19)(20)(21)(22). In this paper, we describe the development of a singlemolecule fluorescence method that probes individual oligomers in a mixture, characterizes their conformations, and determines oligomerization kinetics.…”

Single‐molecule pull‐down (SiMPull) for new‐age biochemistry

“…These numbers can change as a result of drug treatment, mutations , and different cell cycle stages. The effect of mutations has been shown for Ras signaling dynamics in cancer signaling by single‐molecule pull‐down approach . Single complex sensitivity also allows the capture and quantification of low abundance complexes, enabling us to resolve heterogeneous populations.…”

“…They call this method SiMPull, which stands for single‐molecule pull‐down . Two other labs have also successfully utilized a similar approach . In this review, we will discuss the detailed methodology of the assay.…”