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Development and Validation of a Novel Quantitative Assay for Cell surface Expression of GPCRs using a Receptor β-lactamase fusion Protein and the Colourometric Substrate Nitrocefin

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

Identiferoai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/35620
Date12 July 2013
CreatorsLam, Vincent
ContributorsSalahpour, Ali
Source SetsUniversity of Toronto
Languageen_ca
Detected LanguageEnglish
TypeThesis

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