Skip NavigationSkip to Content
Return Return to Search Results

Studying RAS Interactions in Live Cells with BRET

  1. Author:
    Columbus,John
    Turbyville,Tommy

  2. Author Address

    NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA., NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA. turbyvillet@nih.gov.,
    1. Year: 2024
  2. Journal: Methods in Molecular Biology (Clifton, N.J.)
    1. 2797
    2. Pages: 253-260
  4. Type of Article: Article
  1. Abstract:

    Bioluminescence resonance energy transfer (BRET) is a valuable technique for studying protein-protein interactions (PPIs) within live cells (Pfleger and Eidne, Nat Methods 3:165-174, 2006). Among the various BRET methodologies, a recent addition called NanoBRET has emerged, leveraging advancements in donor and acceptor technologies (Machleidt and Woodroofe, ACS Chem Biol 10:1797-1804, 2015). In this study, we present a developed methodology designed to measure PPIs involving the RAS protein family and their effectors and interactors at the plasma membrane. By utilizing the NanoLuc and HaloTag BRET pair, we provide evidence of a saturable interaction between KRAS4b-G12D and full-length RAF1. Conversely, the RAF1 R89L mutant, known to impede RAF1 binding to active RAS, exhibits nonspecific interactions. The assay exhibits remarkable signal-to-background ratios and is highly suitable for investigating the interactions of RAS with effectors, as well as for high-throughput screening assays. © 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

    See More

  1. Keywords:

External Sources

  1. View Full Text
  2. DOI: 10.1007/978-1-0716-3822-4_18
  3. PMID: 38570465

Library Notes

  1. Fiscal Year: FY2023-2024
NCI at Frederick

You are leaving a government website.

This external link provides additional information that is consistent with the intended purpose of this site. The government cannot attest to the accuracy of a non-federal site.

Linking to a non-federal site does not constitute an endorsement by this institution or any of its employees of the sponsors or the information and products presented on the site. You will be subject to the destination site's privacy policy when you follow the link.

ContinueCancel