Hong-Won Lee1,2, Ji Young Ryu1,2, Janghyun Yoo1,2, Byungsan Choi1,2, Kipom Kim1,2 & Tae-Young Yoon1,2
1National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST, Daejeon, South Korea. 2Department of Physics, KAIST, Daejeon, South Korea.
Correspondence should be addressed to Kipom Kim, Tae-Young Yoon
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.