A novel assay for measurement of membrane-protein surface expression using a ß-lactamase.
Traffic
; 14(7): 778-84, 2013 Jul.
Article
em En
| MEDLINE
| ID: mdl-23574269
ABSTRACT
The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one-step and potentially high-throughput assay, using the ß-lactamase enzyme (ßlac) as a reporter, for measurement of surface expression of proteins. In this assay, the ßlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell-impermeable substrate of ßlac, nitrocefin, is used. We demonstrate the utility of the ßlac assay using well-established paradigms of internalization and molecular chaperoning, applied to two G-protein-coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Beta-Lactamases
/
Técnicas de Visualização da Superfície Celular
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Proteínas de Membrana
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article