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

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Zhuang, H; Matsunami, H

Published Date

  • 2008

Published In

Volume / Issue

  • 3 / 9

Start / End Page

  • 1402 - 1413

PubMed ID

  • 18772867

Pubmed Central ID

  • 18772867

Electronic International Standard Serial Number (EISSN)

  • 1750-2799

Digital Object Identifier (DOI)

  • 10.1038/nprot.2008.120

Language

  • eng

Conference Location

  • England