Protocol to measure and analyze protein interactions in mammalian cells using bioluminescence resonance energy transfer
Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here, we present a protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for their preparation for BRET measurements on a CLARIOstar microplate reader and detailed data analysis. For complete details on the use and execution of this protocol, please refer to Steffen et al.<sup>1</sup>.
Authors
- Daniel Abankwa (ORCID: https://orcid.org/0000-0003-2769-0745)
- Candy Laura Steffen (ORCID: https://orcid.org/0000-0002-9445-0074)
- Karolina Pavic (ORCID: https://orcid.org/0000-0002-5958-7438)
- Carla Jane Duval (ORCID: https://orcid.org/0000-0003-2254-3738)
Institutions
- University of Luxembourg (LU)
Publication Details
- Journal
- STAR Protocols
- Published
- 2024-09-28
- DOI
- https://doi.org/10.1016/j.xpro.2024.103348
- Citations
- 5
- Primary Topic
- bioluminescence and chemiluminescence research
- Type
- article
- Field-Weighted Citation Impact
- 1.17
Funders
- UK Research and Innovation
- Fonds National de la Recherche Luxembourg
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek