Yamaguchi, J., Nemoto, N., Sasaki, T., Tokumasu, A., Mimori-Kiyosue, Y., Yagi, T., Funatsu, T. (2001). Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging.  FEBS Lett. 502(3): 79--83.

FBrf0137451

Research paper

Detection of protein-protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein-protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.