A simple method to generate stable cell lines for the analysis of transient protein-protein interactions
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...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2013-04-01
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| Series: | BioTechniques |
| Subjects: | |
| Online Access: | https://www.future-science.com/doi/10.2144/000114013 |
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| Summary: | 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. |
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| ISSN: | 0736-6205 1940-9818 |