Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

70-POS Board 36 A SYSTEMATIC APPROACH FOR THE PURIFICATION OF FLUOROPHORE LABELED PROTEINS VIA ANION EXCHANGE CHROMATOGRAPHY Nicolas D Wendler 1,2 ; Thorben Cordes 1,2 ; 1 Ludwig-Maximilians-Universität München, Faculty of Biology, Planegg-Martinsried, Germany 2 Technische Universität Dortmund, Department of Chemistry and Chemical Biology, Dortmund, Germany Stochastic labeling of proteins with donor and acceptor fluorophores is widely used for Förster resonance energy transfer (FRET) experiments but produces heterogeneous mixtures of donor only, acceptor-only, and donor-acceptor labeled species. Here, we present a strategy for the enrichment of donor-acceptor labeled proteins by combining a simple stochastic labeling protocol with a single step purification using anion exchange (AIEX) chromatography. While this technique has been used before for this purpose, we provide a conceptual basis to systematically design a purification protocol for an arbitrary choice of fluorophores. By characterizing the interaction of hydrolyzed fluorophore-maleimides with the AIEX column material, we are able to rationally select and predict which pairs of fluorophores enable a successful purification of donor-acceptor-labeled protein with yields up to 98%.We demonstrate the capabilities of the method for bulk and single-molecule FRET (smFRET) assays of various bacterial substrate-binding proteins, which are examples of soluble, folded, and well characterized protein systems. The enriched preparations increase the fraction of informative donor-acceptor molecules, reduce acquisition times, and improve the quality of quantitative smFRET analyses. Our results provide practical design principles for fluorophore selection and AIEX purification that facilitate the preparation of high-quality samples for FRET-based biophysical studies.

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