Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.
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.
National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST, Daejeon, South Korea.
Hong-Won
HW
Lee
Ji Young
JY
Ryu
Janghyun
J
Yoo
Byungsan
B
Choi
Kipom
K
Kim
Tae-Young
TY
Yoon
D047468:Immunoprecipitation / Q000379:methods*; D007700:Kinetics; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012984:Software
D011506:Proteins