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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

2.8K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Super-resolution Fluorescence Microscopy01:37

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Related Experiment Video

Updated: Mar 19, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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Time-Resolved Fluorescence Assays.

Chen-Ting Ma1, Eduard A Sergienko2

  • 1Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 19, 2016
PubMed
Summary
This summary is machine-generated.

Time-resolved fluorescence resonance energy transfer (TR-FRET) offers robust, sensitive detection for high throughput screening. This advanced technique minimizes interference, providing a larger assay window for biochemical and cell-based assays.

Keywords:
Enzyme activity assayProduct detectionProtein-protein interactionTR-FRETTime-resolved fluorescence resonance energy transfer

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Area of Science:

  • Biochemistry and Molecular Biology
  • Assay Development
  • High Throughput Screening

Background:

  • Fluorescence-based detection is crucial for sensitive and cost-effective high throughput screening.
  • Existing methods like fluorescence intensity and polarization assays have limitations such as signal interference and narrow assay windows.
  • Fluorescence resonance energy transfer (FRET) is effective for detecting molecular interactions but is constrained by fluorophore proximity.

Purpose of the Study:

  • To highlight the advantages of Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) over other fluorescence-based techniques.
  • To emphasize the performance and robustness of TR-FRET in various assay formats.
  • To showcase the broad applicability of TR-FRET in biochemical and cell-based assays.

Main Methods:

  • Comparison of four common fluorescence-based detection techniques: fluorescence intensity, fluorescence polarization, FRET, and TR-FRET.
  • Focus on the characteristics, advantages, and limitations of each technique, particularly regarding signal interference and assay window.
  • Discussion of TR-FRET's utilization of long-lived fluorescent probes for enhanced signal detection.

Main Results:

  • TR-FRET demonstrates superior performance with a larger assay window and minimized compound spectral interference compared to other methods.
  • TR-FRET overcomes the distance constraints of traditional FRET by using long-lived fluorophores.
  • The technique is versatile, applicable to both biochemical and cell-based in vitro assays.

Conclusions:

  • TR-FRET represents an advanced and robust fluorescence detection method suitable for high throughput screening.
  • Its ability to minimize interference and provide a wider assay window makes it ideal for detecting protein-protein interactions, biochemical reaction products, and cellular activities.
  • TR-FRET is a valuable tool for modern drug discovery and biological research.