Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging.
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.
Yamaguchi
J
J
Department of Physics, School of Science and Engineering, Waseda University, Tokyo, Japan.
Nemoto
N
N
Sasaki
T
T
Tokumasu
A
A
Mimori-Kiyosue
Y
Y
Yagi
T
T
Funatsu
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary; D004330:Drosophila / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis*; D007091:Image Processing, Computer-Assisted; D016547:Kinesins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008870:Microtubules / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D013552:Swine; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D016547:Kinesins