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Updated: May 4, 2026

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells
Published on: August 31, 2011
Labeling thiols on proteins, living cells, and tissues with enhanced emission induced by FRET
Yue Yuan1, Xijun Wang2, Bin Mei1
1CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China.
Abstract:
Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved.
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