A simple method to generate stable cell lines for the analysis of transient protein-protein interactions
| Autor: | Emilia Elizabeth Savage, Denise Wootten, Arthur Christopoulos, Patrick Michael Sexton, Sebastian George Barton Furness |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 54, Iss 4, Pp 217-221 (2013) |
| Druh dokumentu: | article |
| ISSN: | 00011401 1940-9818 0736-6205 |
| DOI: | 10.2144/000114013 |
| Popis: | Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energ y transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins. |
| Databáze: | Directory of Open Access Journals |
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