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Related Experiment Videos

Detection of programmed cell death using fluorescence energy transfer

X Xu1, A L Gerard, B C Huang

  • 1Rigel, Inc., 772 Lucerne Drive, Sunnyvale, CA 94086, USA.

Nucleic Acids Research
|June 6, 1998
PubMed
Summary

This study links green and blue fluorescent proteins using a peptide cleaved by Caspase-3. Protease activity monitoring via fluorescence energy transfer (FRET) can track apoptosis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Fluorescence energy transfer (FRET) occurs when linked fluorescent proteins like green fluorescent protein (GFP) and blue fluorescent protein (BFP) are close.
  • Protease activity can disrupt this linkage, thereby eliminating the FRET effect.
  • Caspase-3 (CPP32) is a key protease activated during programmed cell death (apoptosis).

Purpose of the Study:

  • To develop a FRET-based assay for monitoring Caspase-3 activation during apoptosis.
  • To create a biosensor utilizing the FRET principle and protease-specific cleavage.

Main Methods:

  • Covalently linking GFP and BFP via an 18-amino acid peptide containing the Caspase-3 recognition sequence (DEVD).
  • Utilizing FRET measurements to detect the cleavage of the peptide linkage.

Related Experiment Videos

  • Monitoring FRET changes in real-time to assess Caspase-3 activity.

    • Main Results:

    • The linked GFP-BFP construct exhibited FRET when the peptide linker was intact.
    • Cleavage of the DEVD sequence by activated Caspase-3 resulted in the loss of FRET.
    • The FRET assay effectively indicated Caspase-3 activation during the apoptosis process.

    Conclusions:

    • A novel FRET-based biosensor was successfully developed to monitor Caspase-3 activity.
    • This method provides a sensitive and real-time approach to study apoptosis.
    • The FRET assay offers a valuable tool for investigating cellular protease function in biological processes.