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Peer-reviewedCC BYThe Analyst

Characterization of membrane protein-nanobody complexation via flow-induced dispersion analysis (FIDA).

  • Kiefer O Ramberg1,2
  • Monika Olejnik3,4
  • Saúl Gallego-Martínez2
  • Andrew Quigley4
  • Martin Caffrey2
4 affiliations
  1. 1School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, D09 V209, Ireland. [email protected].
  2. 2School of Biochemistry & Immunology, Trinity College Dublin, Dublin, D02 R590, Ireland. [email protected].
  3. 3Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, UK.
  4. 4Membrane Protein Laboratory, Diamond Light Source, Research Complex at Harwell, Didcot, UK.
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Abstract

Nanobody binders may be raised against membrane protein targets for a myriad of applications including the modulation of bioactivity and to aid in structural elucidation. Flow-Induced Dispersion Analysis (FIDA) is a burgeoning analytical technique which has been used to quantify protein-protein binding interactions. FIDA offers a straightforward route to characterizing membrane protein-nanobody complexation in terms of affinity and particle size. A protocol for the rapid characterisation of nanobodies that bind membrane proteins with high affinity using FIDA would expedite the development of novel nanobody-based biomedicines and facilitate nanobody-assisted membrane protein structural biology. In this study, FIDA was used to characterize the complexation of a nanobody and a model membrane integral protein transporter. To this end, the nanobody was fluorescently labelled using fluorescein isothiocyanate. A simple two-step protocol for producing labelled nanobody stocks devoid of interfering free dye is presented. FIDA experiments entailing sequential addition of the micellarised transporter protein to the labelled nanobody in detergent-containing buffer revealed single digit nanomolar binding consistent with binding constants obtained previously by surface plasmon resonance analysis.

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Cite

BibTeXRIS
BibTeX
@article{ramberg2026characterization,
  title = {{Characterization of membrane protein-nanobody complexation via flow-induced dispersion analysis (FIDA).}},
  author = {Kiefer O Ramberg and Monika Olejnik and Saúl Gallego-Martínez and Andrew Quigley and Martin Caffrey},
  journal = {The Analyst},
  year = {2026},
  month = oct,
  doi = {10.1039/d6an01036a},
  url = {https://europepmc.org/article/MED/42968699},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Characterization of membrane protein-nanobody complexation via flow-induced dispersion analysis (FIDA).
AU  - Kiefer O Ramberg
AU  - Monika Olejnik
AU  - Saúl Gallego-Martínez
AU  - Andrew Quigley
AU  - Martin Caffrey
PY  - 2026
DA  - 2026/10/29
JO  - The Analyst
DO  - 10.1039/d6an01036a
UR  - https://europepmc.org/article/MED/42968699
AB  - Nanobody binders may be raised against membrane protein targets for a myriad of applications including the modulation of bioactivity and to aid in structural elucidation. Flow-Induced Dispersion Analysis (FIDA) is a burgeoning analytical technique which has been used to quantify protein-protein binding interactions. FIDA offers a straightforward route to characterizing membrane protein-nanobody complexation in terms of affinity and particle size. A protocol for the rapid characterisation of nanobodies that bind membrane proteins with high affinity using FIDA would expedite the development of novel nanobody-based biomedicines and facilitate nanobody-assisted membrane protein structural biology. In this study, FIDA was used to characterize the complexation of a nanobody and a model membrane integral protein transporter. To this end, the nanobody was fluorescently labelled using fluorescein isothiocyanate. A simple two-step protocol for producing labelled nanobody stocks devoid of interfering free dye is presented. FIDA experiments entailing sequential addition of the micellarised transporter protein to the labelled nanobody in detergent-containing buffer revealed single digit nanomolar binding consistent with binding constants obtained previously by surface plasmon resonance analysis.
SN  - 0003-2654
SN  - 1364-5528
ER  -