Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity   

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Tie Liu
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Original research article

A brief version of this protocol appeared in:
Mar 2017


Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties.

In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS). We have successfully used this method for identification and characterization of transporters of the plant defense metabolites called glucosinolates and cyanogenic glucosides (Jørgensen et al., 2017), however the method is usable for the characterization of any transporter whose substrate can be detected by LC-MS.

Keywords: Xenopus laevis oocytes, Uptake assays, Transporter characterization, Liquid chromatography-mass spectrometry


Oocytes from the African clawed frog (Xenopus laevis) is a well-established expression system for heterologous expression and characterization of membrane proteins (i.e., transporters and channels). The X. laevis oocyte express few endogenous membrane proteins and has a low background transport activity. Furthermore, secondary active transporters from plants (Boorer et al., 1992; Theodoulou and Miller, 1995; Nour-Eldin et al., 2006), animals (Sumikawa et al., 1981; Sigel, 1990) and microbes (Calamita et al., 1995; Wahl et al., 2010) have been successfully expressed in X. laevis oocytes, showing that this system is widely applicable to characterize transporters from any organism.

A transport assay requires the expression of the transport protein in a system capable of folding the protein correctly and localizing it to a membrane across which movement of substrate can be detected. Due to the minute amounts moved, researchers have typically used radiolabeled substrates for transport assays. By washing oocytes after incubation and scintillation counting of the oocytes interior accumulation of substrate inside the oocyte could be detected. We have previously utilized this method to identify and characterize sucrose and glucose transporters from Arabidopsis thaliana using the Xenopus oocytes system (Nour-Eldin et al., 2006; Norholm et al., 2006). However, for identification and characterization of plant specialized metabolite transporters, it can be very challenging to generate radiolabeled isotopes of a target substrate. To overcome this challenge we developed a protocol for detecting and quantifying transport of specialized metabolites into X. laevis oocytes by use of LC-MS. Use of this method has allowed us to expand the inventory of assayable substrates to anything that can be detected and quantified by the LC-MS system applied.

Copyright Jørgensen et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jørgensen, M. E., Crocoll, C., Halkier, B. A. and Nour-Eldin, H. H. (2017). Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity. Bio-protocol 7(20): e2581. DOI: 10.21769/BioProtoc.2581.
  2. Jørgensen, M. E. Xu, D., Crocoll, C., Ramírez, D., Motawia, M. S., Olsen, C. E., Nour-Eldin, H. H. and Halkier, B. A. (2017). Origin and evolution of transporter substrate specificity within the NPF family. eLife 6: e19466.

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