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Rapid functional analysis of protein–protein interactions by fluorescent C-terminal labeling and single-molecule imaging

Authors
Journal
FEBS Letters
0014-5793
Publisher
Wiley Blackwell (John Wiley & Sons)
Publication Date
Volume
502
Issue
3
Identifiers
DOI: 10.1016/s0014-5793(01)02581-9
Keywords
  • Protein–Protein Interaction
  • Single-Molecule Imaging
  • Protein Labeling
  • Puromycin
  • Fluorescence Microscopy
  • Cell-Free Translation
Disciplines
  • Biology

Abstract

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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