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Related Experiment Video

Updated: May 7, 2026

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
10:26

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Published on: November 7, 2019

Visualization of ubiquitin conjugates using ubiquitin-mediated fluorescence complementation analysis.

Tom K Kerppola

    Cold Spring Harbor Protocols
    |October 3, 2013
    PubMed
    Summary
    This summary is machine-generated.

    Ubiquitin conjugation modifies protein function and stability. A new assay visualizes these ubiquitin conjugates in live cells using fluorescence complementation, overcoming limitations of traditional methods.

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    Last Updated: May 7, 2026

    Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
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    Published on: November 7, 2019

    Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
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    Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain
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    Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

    Published on: May 21, 2019

    Area of Science:

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Background:

    • Ubiquitin-family peptide conjugation is a crucial post-translational modification regulating protein function and stability.
    • This modification involves attaching ubiquitin peptides to lysine residues on substrate proteins, offering diverse regulatory possibilities.
    • Current detection methods like immunoprecipitation and immunoblotting are unsuitable for live-cell imaging or determining subcellular localization.

    Purpose of the Study:

    • To develop a novel method for visualizing ubiquitin conjugates in live cells.
    • To overcome the limitations of existing biochemical assays in dynamic cellular contexts.
    • To enable the study of ubiquitin conjugation dynamics and localization within living cells.

    Main Methods:

    • Development of a ubiquitin-mediated fluorescence complementation assay (UMFCA).
    • Fusion of ubiquitin to one fragment of a fluorescent protein and the substrate protein to a complementary fragment.
    • Observation of fluorescence complementation upon ubiquitin conjugation in cultured mammalian cells.

    Main Results:

    • Successful visualization of ubiquitin conjugates in live mammalian cells using UMFCA.
    • Demonstration of the assay's ability to track ubiquitin conjugation events in real-time.
    • Establishment of a method adaptable to various cell types and organisms.

    Conclusions:

    • The ubiquitin-mediated fluorescence complementation assay provides a powerful tool for live-cell imaging of ubiquitin conjugation.
    • This technique overcomes the limitations of traditional methods, enabling detailed studies of ubiquitin signaling.
    • UMFCA is a versatile assay applicable across diverse biological systems for investigating protein ubiquitination.