Protocol to measure and analyze protein interactions in mammalian cells using bioluminescence resonance energy transfer
Year: 2024
Type: article
Abstract: 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. 1 • Cell-based protein-protein interaction quantification protocol using BRET • Sensitivity even for transient interactions in K-RasG12V nanocluster • Comprehensive instructions for setup and analysis of BRET experiments Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Source: STAR Protocols
Institution: University of Luxembourg
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
Awards: G2034880924, G4357971614, G4669496973
OpenAlex ID: W4402945878