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Related Concept Videos

Labeling DNA Probes03:31

Labeling DNA Probes

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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Detection of Protease Activity by Fluorescent Peptide Zymography
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Published on: January 20, 2019

Genetically encoded protease substrate based on lanthanide-binding peptide for time-gated fluorescence detection.

Johanna Vuojola1, Markku Syrjänpää, Urpo Lamminmäki

  • 1Department of Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland. johanna.vuojola@utu.fi

Analytical Chemistry
|January 1, 2013
PubMed
Summary

This study introduces a novel FRET assay for protease activity using lanthanide labels and green fluorescent protein. This genetically encoded system offers a sensitive and versatile method for detecting caspase-3 and other proteases.

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Area of Science:

  • Biomolecular interactions
  • Biomedical research
  • Biochemistry

Background:

  • Fluorescence and Förster resonance energy transfer (FRET) are crucial for studying biomolecular interactions.
  • Fluorescent proteins allow genetic encoding for in vivo and recombinant protein applications.
  • Lanthanide labels offer advantages over fluorescent proteins by reducing autofluorescence and enabling time-gating for enhanced assay sensitivity.

Purpose of the Study:

  • To develop a FRET-based homogeneous protease activity assay.
  • To combine terbium-ion-containing lanthanide-binding peptide (Tb(3+)-LBP) with green fluorescent protein (GFP) for protease detection.
  • To demonstrate the applicability of a Tb(3+)-LBP-GFP energy-transfer pair in a protease activity assay.

Main Methods:

  • A genetically engineered construct was designed with adjacent LBP and GFP sequences linked by a caspase-3 recognition sequence.
  • A FRET-based homogeneous protease activity assay was developed using this construct.
  • The assay was applied for the detection of caspase-3 activity.

Main Results:

  • The study successfully demonstrated the applicability of a Tb(3+)-LBP-GFP energy-transfer pair in a protease activity assay.
  • The genetically encodable and intrinsically fluorescent components allowed for easy expression without chemical labeling.
  • The assay showed potential for detecting caspase-3 activity.

Conclusions:

  • The developed Tb(3+)-LBP-GFP FRET system provides a sensitive and versatile method for protease activity detection.
  • The genetically encoded nature of the components simplifies assay development and application.
  • The method can be adapted for detecting various proteases by modifying the fluorescent protein and linker sequence.