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Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay

Abstract

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[3H]arginine or D-[3H]glucose) to acceptor proteins immobilized on fluoromicrospheres (containing the scintillant), a light signal is stimulated and measured. The application of SPA to purified, detergent-solubilized membrane transport proteins allows substrate-binding properties to be assessed (e.g., substrate specificity and affinity), usually within 1 d. Notably, the SPA makes it possible to study specific transporters without interference from other cellular components, such as endogenous transporters. Reconstitution of the target transporter into proteoliposomes is not required. The SPA procedure allows high sample throughput and simple sample handling without the need for washing or separation steps: components are mixed in one well and the signal is measured directly after incubation. Therefore, the SPA is an excellent tool for high-throughput screening experiments, e.g., to search for substrates and inhibitors, and it has also recently become an attractive tool for drug discovery.

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Figure 1: Principle of the SPA.
Figure 2: Optimization of the SPA signal illustrated for AdiC using L-[3H]arginine and PVT beads.
Figure 3: SPA signal validation.
Figure 4: Possible applications of the SPA for transporter characterization.

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Acknowledgements

We are grateful to M. Quick for fruitful discussions, M. Palacin and Z. Ucurum for providing the AdiC constructs, J.-M. Jeckelmann for the IICB clone and J. Gertsch for access to the scintillation counters. This work was supported by the Swiss National Foundation for Scientific Research (grants 31003A_125150 and 31SY30-131038 to D.F.), the European Science Foundation (grant 09-EuroSYNBIO-FP-012 NANOCELL to D.F.), the Novartis Foundation and the National Centre of Competence in Research (NCCR) TransCure.

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D.H. designed and performed experiments, analyzed data and wrote the manuscript; D.F. designed experiments, analyzed data and wrote the manuscript.

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Correspondence to Dimitrios Fotiadis.

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Harder, D., Fotiadis, D. Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay. Nat Protoc 7, 1569–1578 (2012). https://doi.org/10.1038/nprot.2012.090

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