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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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...
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.
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Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...

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Related Experiment Video

Updated: May 18, 2026

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level
08:29

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Published on: April 19, 2019

Labeling proteins for single-molecule FRET.

Chirlmin Joo, Taekjip Ha

    Cold Spring Harbor Protocols
    |September 6, 2012
    PubMed
    Summary

    This study details a method for labeling proteins for single-molecule Förster resonance energy transfer (smFRET) using total internal reflection microscopy. This technique enables precise distance measurements in biological studies.

    Area of Science:

    • Biophysics
    • Molecular Biology
    • Microscopy

    Background:

    • Single-molecule (sm) fluorescence detection offers advantages over ensemble methods by avoiding time and population averaging.
    • Förster (fluorescence) resonance energy transfer (FRET) is a biophysical technique used to measure distances between 30-80 Å.
    • Total internal reflection (TIR) microscopy is a variant of smFRET that enhances sensitivity.

    Purpose of the Study:

    • To describe a protocol for labeling proteins for single-molecule Förster resonance energy transfer (smFRET) experiments.
    • To adapt smFRET techniques for use with total internal reflection (TIR) microscopy.
    • To provide a specific example using E. coli Rep helicase.

    Main Methods:

    • Protein labeling strategies for smFRET.

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  • Implementation of total internal reflection (TIR) microscopy for fluorescence detection.
  • Application of Förster resonance energy transfer (FRET) principles.

    • Main Results:

    • A detailed protocol for labeling proteins for smFRET with TIR microscopy is presented.
    • The protocol is demonstrated using E. coli Rep helicase.
    • The method allows for sensitive detection of molecular interactions and conformational changes.

    Conclusions:

    • The described protein labeling protocol is effective for smFRET studies using TIR microscopy.
    • This method provides a valuable tool for investigating molecular mechanisms at the single-molecule level.
    • Adaptations may be necessary for different proteins or experimental conditions.