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

  1. Author:
    Smith,Russell
    Morrison,Deborah

  2. Author Address

    Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702, USA. Electronic address: smithrs@mail.nih.gov., Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702, USA. Electronic address: morrisod@mail.nih.gov.,
    1. Year: 2023
    2. Date: Aug 16
    3. Epub Date: 2023 08 16
  2. Journal: STAR Protocols
    1. 4
    2. 3
    3. Pages: 102461
  4. Type of Article: Article
  5. Article Number: 102461
  1. Abstract:

    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. Published by Elsevier Inc.

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External Sources

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  2. DOI: 10.1016/j.xpro.2023.102461
  3. PMID: 37590148
  4. PMCID: PMC10440347
  5. PII : S2666-1667(23)00428-8

Library Notes

  1. Fiscal Year: FY2022-2023
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