Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

08 January 2020, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. Here we describe the practical details of a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.


Ion-exchange chromatography
Fluorescent Dye
Fluorescence spectroscopy
Förster resonance energy transfer
Intrinsically disordered proteins
Protein Folding
Single-molecule spectroscopy


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