Protocol for measuring BRAF autoinhibition in live cells using a proximity-based NanoBRET assay

BRAF is frequently activated via mutation in human cancer and the RASopathy syndromes; however, for BRAF activation to occur, autoinhibitory interactions between the regulatory and catalytic domains must be relieved. Here, we present a proximity-based NanoBRET (bioluminescence resonance energy trans...

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Detalles Bibliográficos
Autores principales: Spencer-Smith, Russell, Morrison, Deborah K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10440347/
https://www.ncbi.nlm.nih.gov/pubmed/37590148
http://dx.doi.org/10.1016/j.xpro.2023.102461
Descripción
Sumario:BRAF is frequently activated via mutation in human cancer and the RASopathy syndromes; however, for BRAF activation to occur, autoinhibitory interactions between the regulatory and catalytic domains must be relieved. Here, we present a proximity-based NanoBRET (bioluminescence resonance energy transfer) assay for real-time measurement of BRAF autoinhibition in live cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for reading the NanoBRET emissions and confirming protein expression. For complete details on the use and execution of this protocol, please refer to Spencer-Smith et al. (2022).(1)

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