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bioRxiv · 10.64898/2025.12.30.697019

A systematic approach for the purification of fluorophore-labelled proteins via anion exchange chromatography

Wendler, N.·Cordes, T.
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Abstract

Forster resonance energy transfer (FRET), an optical distance ruler, provides the unique ability to monitor molecular interactions and conformational changes in single biomacromolecules and multi-subunit complexes. It has proven to be a powerful tool to study enzymatic reactions, membrane transport, protein folding, but also the properties of nucleic acids, proteins, molecular motors and many other biological systems and processes. A prerequisite for FRET is targeted (covalent) labelling of macromolecules with two distinct fluorophores. Here, we present a strategy for stochastic labeling of protein residues with the required donor and acceptor dyes via anion exchange 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 fluorophore-maleimides with the column material, we are able to select (and predict) which pairs of fluorophores allow successful purification of donor-acceptor-labelled protein with yields up to 98%. We demonstrate the capabilities of the method for bulk and single-molecule FRET assays of various bacterial substrate-binding proteins.

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Wendler, N., Cordes, T.. 2025-12-30. A systematic approach for the purification of fluorophore-labelled proteins via anion exchange chromatography. https://doi.org/10.64898/2025.12.30.697019

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