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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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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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High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
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Quantifying transcription factor binding dynamics at the single-molecule level in live cells.

Diego M Presman1, David A Ball1, Ville Paakinaho1

  • 1Laboratory of Receptor Biology and Gene Expression, Building 41, 41 Library Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Methods (San Diego, Calif.)
|March 19, 2017
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Summary

Single-molecule tracking (SMT) in living cells reveals transcription factor binding dynamics. This study details methods for analyzing protein residence time and bound fractions, advancing cell biology research.

Keywords:
DNA bindingDynamicsFluorescence microscopyGlucocorticoid receptorSingle-molecule trackingTranscription factor

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Quantitative fluorescence microscopy enables studying dynamic cellular processes.
  • Single-molecule studies in living cells offer new insights into molecular mechanisms.
  • Lack of consensus on image acquisition and data analysis hinders single-molecule tracking (SMT).

Purpose of the Study:

  • To provide a detailed approach for single-molecule tracking (SMT) of transcription factors in living cells.
  • To determine key binding characteristics like residence time and bound fractions.
  • To compare different labeling strategies and data analysis methods for SMT.

Main Methods:

  • Utilizing quantitative fluorescence microscopy and advanced imaging techniques.
  • Performing single-molecule tracking (SMT) on transcription factors within living cells.
  • Comparing various fluorophores, labeling densities, and microscopy setups.
  • Analyzing data using established and novel methods to extract kinetic parameters.

Main Results:

  • Demonstrated a robust method for SMT of transcription factors.
  • Evaluated different protein tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for multicolor applications.
  • Compared methods for extracting dissociation rates using simulated and experimental data.
  • Provided insights into optimal conditions for SMT data acquisition and analysis.

Conclusions:

  • Established a comprehensive protocol for single-molecule tracking of transcription factors.
  • Highlighted the importance of careful selection of fluorophores, tags, and analysis methods.
  • Addressed current challenges and future directions in single-molecule studies of cellular dynamics.