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Protocol for measuring BRAF autoinhibition in live cells using a proximity-based NanoBRET assay
Russell Spencer-Smith1, Deborah K Morrison1
1Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702, USA.
STAR Protocols
|August 17, 2023
Summary
Researchers developed a novel NanoBRET assay to measure BRAF autoinhibition in real-time within live cells. This method is crucial for understanding BRAF activation in cancer and RASopathy syndromes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- BRAF mutations are common in human cancers and RASopathy syndromes.
- BRAF activation requires relieving autoinhibitory interactions between its regulatory and catalytic domains.
Purpose of the Study:
- To develop and present a proximity-based NanoBRET assay for real-time measurement of BRAF autoinhibition in live cells.
- To provide a detailed protocol for utilizing this assay.
Main Methods:
- Utilized a proximity-based NanoBRET (bioluminescence resonance energy transfer) assay.
- Detailed procedures for cell seeding, transfection, replating, and NanoBRET emission reading.
- Confirmed protein expression post-assay.
Main Results:
- Successfully established a NanoBRET assay for real-time monitoring of BRAF autoinhibition.
- Demonstrated the protocol for cell handling and data acquisition using the assay.
Conclusions:
- The developed NanoBRET assay offers a novel tool for studying BRAF autoinhibition dynamics in live cells.
- This assay facilitates research into BRAF-related diseases, including cancer and RASopathies.
