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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...
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...
Immunogold Electron Microscopy01:20

Immunogold Electron Microscopy

Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.

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Related Experiment Video

Updated: Jun 16, 2026

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Optimizing multifunctional fluorescent ligands for intracellular labeling.

Pratik Kumar1, Jason D Vevea2,3,4, Ariana N Tkachuk1

  • 1Janelia Research Campus, HHMI, Ashburn, VA 20147.

Proceedings of the National Academy of Sciences of the United States of America
|October 27, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed cell-permeable fluorescent tags for labeling proteins in living cells. These new tools allow for protein purification and targeted nuclear translocation, enhancing cellular research capabilities.

Keywords:
HaloTagchemical biologychemistryfluorescenceprotein purification

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Area of Science:

  • Chemical Biology
  • Molecular Biology
  • Medicinal Chemistry

Background:

  • Enzyme-based self-labeling tags facilitate covalent attachment of synthetic molecules to proteins within living cells.
  • Designing cell-permeable multifunctional ligands with fluorophores, affinity tags, or pharmacological agents is challenging due to potential membrane permeability issues.

Purpose of the Study:

  • To investigate the chemical properties of rhodamine-based self-labeling tag ligands to improve cell permeability.
  • To design and validate cell-permeable multifunctional ligands for intracellular protein manipulation.

Main Methods:

  • Medicinal chemistry analysis of rhodamine-based ligands, focusing on lactone-zwitterion equilibrium constant (K_L-Z) and distribution coefficients (logD_7.4).
  • Design and synthesis of multifunctional HaloTag ligands incorporating biotin or JQ1 moieties.
  • Cellular experiments to assess ligand-mediated protein translocation and transcriptional activity.

Main Results:

  • A negative correlation was observed between K_L-Z and logD_7.4, indicating that low K_L-Z and high logD_7.4 dyes (e.g., Si-rhodamines) enhance cell entry.
  • Successfully designed cell-permeable HaloTag ligands for mitochondrial purification (biotin) and nuclear translocation of BRD4 (JQ1).
  • Translocation of BRD4 to constitutive heterochromatin resulted in increased transcriptional activity.

Conclusions:

  • The study provides design principles for creating cell-permeable, multifunctional fluorescent ligands.
  • These novel reagents enable affinity capture and targeted translocation of intracellular proteins in living cells.
  • The developed chemical tools will advance biological research by enabling precise manipulation of cellular components.