Cell Biology; Cell-based Assays; Molecular Biology; Neuroscience (all); Biochemistry, Genetics and Molecular Biology (all); Immunology and Microbiology (all)
Résumé :
[en] 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.
Disciplines :
Biochimie, biophysique & biologie moléculaire
Auteur, co-auteur :
DUVAL, Carla ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
STEFFEN, Candy ; University of Luxembourg > Faculty of Science, Technology and Medicine > Department of Life Sciences and Medicine > Team Daniel ABANKWA
PAVIC, Karolina ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
ABANKWA, Daniel ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
Co-auteurs externes :
no
Langue du document :
Anglais
Titre :
Protocol to measure and analyze protein interactions in mammalian cells using bioluminescence resonance energy transfer.
This work was supported by grants from the National Research Fund Luxembourg (FNR) INTER/UKRI/19/14174764-RAS-NANOME and INTER/NWO/19/14061736 - HRAS-PPi to D.K.A. and AFR/17927850/Duval C./KRuptor to C.J.D. We are grateful to Dr. Ganesh babu Manoharan for implementing the BRET technology in the Abankwa lab. The graphical abstract was created using BioRender.com .
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