Work

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

Year: 2026

Type: article

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.

Source: The Analyst

DOI: 10.1039/d6an01036a

Authors: Kiefer O. Ramberg, Monika Olejnik, Saúl Gallego-Martínez, Andrew Quigley, Martin Caffrey

Institutions: Trinity College Dublin, Dublin City University, University of Leeds, Diamond Light Source, Research Complex at Harwell

Language: English

Topic: Monoclonal and Polyclonal Antibodies Research

Subfield: Immunology

Field: Immunology and Microbiology

Domain: Life Sciences

Open Access status: hybrid

Funders: Wellcome Trust, Diamond Light Source, Research Ireland

Awards: A membrane protein laboratory for integrative structural biology, A membrane protein laboratory for integrative structural biology