Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging |
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Authors: | Yamaguchi J Nemoto N Sasaki T Tokumasu A Mimori-Kiyosue Y Yagi T Funatsu T |
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Institution: | Department of Physics, School of Science and Engineering, Waseda University, Tokyo, Japan. |
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Abstract: | 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. |
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