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Nat Protoc. 2008;3(9):1402-13. doi: 10.1038/nprot.2008.120.

Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.

Author information

1
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA. hanyi.zhuang@duke.edu

Abstract

A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant-OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.

PMID:
18772867
PMCID:
PMC2664838
DOI:
10.1038/nprot.2008.120
[Indexed for MEDLINE]
Free PMC Article

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