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Related Concept Videos

Labeling DNA Probes03:31

Labeling DNA Probes

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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Related Experiment Video

Updated: Mar 3, 2026

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology
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Activity-Based Probes for HECT E3 Ubiquitin Ligases.

Robert Byrne1, Thomas Mund2, Julien D F Licchesi1

  • 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.

Chembiochem : a European Journal of Chemical Biology
|April 21, 2017
PubMed
Summary

Activity-based probes (ABPs) now effectively label HECT E3 ubiquitin ligases, expanding their utility beyond deubiquitinases. This study demonstrates ABPs can target catalytic HECT domains in vitro and in cells.

Keywords:
HECT E3 ubiquitin ligaseactivity-based probesbiological activityproteasomeprotein-protein interactionsubiquitin

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chemical Biology

Background:

  • Activity-based probes (ABPs) are crucial for studying enzyme function.
  • Their application in investigating cysteine-based E3 ubiquitin ligases, particularly HECT E3 ligases, has been limited.
  • Deubiquitinase research extensively utilizes ABPs, but their potential for E3 ligase studies remains underexplored.

Purpose of the Study:

  • To evaluate the efficacy of existing ubiquitin-ABPs (Ub-VME, Ub-PA) and novel E2-Ub-ABPs for studying HECT E3 ubiquitin ligases.
  • To determine if ABPs can label the catalytic HECT domains of various HECT E3 ligases.
  • To demonstrate the in-cell labeling capability of ABPs on endogenous HECT E3 ligases.

Main Methods:

  • In vitro biochemical assays using ubiquitin-ABPs (Ub-VME, Ub-PA) and E2-Ub-ABPs.
  • Testing ABPs on a panel of purified HECT E3 ubiquitin ligases.
  • Cellular assays using cell lysates to assess labeling of endogenous UBE3C under basal conditions.

Main Results:

  • Ubiquitin-ABPs successfully labeled HECT domains in vitro.
  • E2-Ub-ABPs were shown to label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1, in addition to Parkin.
  • Endogenous UBE3C was labeled by Ub-PA and His-UBE2D2-Ub-ABP in cell lysates.

Conclusions:

  • Activity-based probes can effectively label HECT E3 ubiquitin ligases, expanding their utility.
  • This study establishes a new methodology for investigating HECT E3 ligase activity and function using ABPs.
  • The findings offer valuable tools and insights for future research into HECT E3 ubiquitin ligase biology.