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

Harder, Daniel; Fotiadis, Dimitrios (2012). Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay. Nature protocols, 7(9), pp. 1569-78. London: Nature Publishing Group 10.1038/nprot.2012.090

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Harder, Daniel and Fotiadis, Dimitrios José

ISSN:

1754-2189

Publisher:

Nature Publishing Group

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 14:37

Last Modified:

06 Dec 2013 13:37

Publisher DOI:

10.1038/nprot.2012.090

PubMed ID:

22864198

Web of Science ID:

000308526300001

URI:

https://boris.unibe.ch/id/eprint/15047 (FactScience: 222201)

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