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Nat Protoc. 2012 Sep;7(9):1569-78. doi: 10.1038/nprot.2012.090. Epub 2012 Aug 2.

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

Author information

1
Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.

Abstract

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[(3)H]arginine or D-[(3)H]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.

PMID:
22864198
DOI:
10.1038/nprot.2012.090
[Indexed for MEDLINE]

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