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Rapid functional analysis of protein–protein interactions by fluorescent C‐terminal labeling and single‐molecule imaging
Author(s) -
Yamaguchi Junichi,
Nemoto Naoto,
Sasaki Toru,
Tokumasu Ako,
Mimori-Kiyosue Yuko,
Yagi Toshiki,
Funatsu Takashi
Publication year - 2001
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(01)02581-9
Subject(s) - puromycin , kinesin , biophysics , fluorescence , total internal reflection fluorescence microscope , protein–protein interaction , chemistry , microtubule , motor protein , green fluorescent protein , molecule , fluorescence microscope , translation (biology) , microbiology and biotechnology , protein biosynthesis , biochemistry , messenger rna , biology , gene , physics , organic chemistry , quantum mechanics , membrane
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.