Work

A Systematic Approach for the Purification of Fluorophore‐Labeled Proteins via Anion Exchange Chromatography

Year: 2026

Type: article

Abstract: Förster 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) labeling 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 of hydrolyzed fluorophore-maleimides with the column material, we are able to select (and predict) which pairs of fluorophores allow 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 assays of various bacterial substrate-binding proteins, which are examples of soluble, folded, and well-characterized protein systems.

Source: ChemBioChem

DOI: 10.1002/cbic.70394

Authors: Nicolas D. Wendler, Thorben Cordes

Institutions: TU Dortmund University, Ludwig-Maximilians-Universität München

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,430

Funders: Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung