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Step-by-Step Protocol for Fluorescence-Based Analysis of Uptake in Transporter-Expressing Xenopus laevis Oocytes
Víctor de Prado Parralejo1, Hussam H Nour-Eldin1, Christa Kanstrup1
1Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Abstract:
Xenopus laevis oocytes are widely used as a heterologous expression system for investigating the function of membrane proteins due to robust expression of heterologous protein and a low endogenous transport background. Traditionally, transporter activity in oocytes has been assessed using electrophysiology or radiolabeled uptake assays, approaches that are constrained by the requirement for electrogenicity of the transport process, availability of radiolabeled compounds, and instrumentation. Here, we describe a fluorescence-based uptake assay that enables direct and rapid quantification of transporter activity using a fluorescence plate reader. The protocol uses the Arabidopsis thaliana sucrose transporter 1 (SUC1) and its fluorescent substrate esculin as a case for how to set up the assay. The workflow includes optimizing assay conditions, sample preparation, fluorescent measurements, and downstream data analysis using R. This method can readily be adapted to other transporter-substrate pairs, and it supports applications such as transporter inhibitor screening, mutational analysis, characterization of kinetic properties, or indirect substrate specificity testing through competition assays. Overall, this protocol provides a simple and scalable alternative to traditional techniques, eliminating the need for radiolabeled compounds or electrophysiology while enabling easy quantitative assessment of transporter activity. Key features • Provides a simple, plate reader-based fluorescence uptake assay for quantifying transporter activity in Xenopus laevis oocytes. • Adaptable to any transporter with a compatible fluorescent substrate, supporting rapid testing of substrate specificity indirectly through competition assays or mutant screening. • Includes a complete workflow from assay setup and conduction to data analysis, suitable for both initial characterization and routine functional testing of membrane transporters.