Rapid functional analysis of protein–protein interactions by fluorescent C‐terminal labeling and single‐molecule imaging
Open Access
- 1 August 2001
- journal article
- Published by Wiley in FEBS Letters
- Vol. 502 (3) , 79-83
- https://doi.org/10.1016/s0014-5793(01)02581-9
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.Keywords
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