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Protein Dynamics in Living Cells

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...

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Updated: Nov 7, 2025

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

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Published on: January 16, 2019

Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide.

Thai Pham¹, Renjie Liao¹, Joshua Labaer¹

¹Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA.

Molecules (Basel, Switzerland)

|April 30, 2021

Summary

This summary is machine-generated.

This study introduces a sensitive protein in situ profiling method using cleavable fluorescent tyramide (CFT) for multiplexed quantification in single cells. The approach accurately measures protein expression heterogeneity and correlations, even in formalin-fixed paraffin-embedded tissues.

Keywords:

heterogeneity immunofluorescence immunohistochemistry proteomics single-cell

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Published on: April 7, 2023

Area of Science:

Cellular Biology
Molecular Biology
Biochemistry

Background:

Understanding complex cellular systems necessitates tools for quantifying protein expression within their native context.
Current methods face limitations in sensitivity, accuracy, and multiplexing capabilities for in situ protein analysis.

Purpose of the Study:

To develop a highly sensitive and accurate protein in situ profiling approach for multiplexed quantification in single cells.
To enable the study of protein expression heterogeneity and correlations in various cellular contexts, including FFPE tissues.

Main Methods:

Utilized off-the-shelf antibodies conjugated with horseradish peroxidase (HRP) and a novel cleavable fluorescent tyramide (CFT).
Implemented iterative cycles of antibody staining, fluorescence imaging, CFT cleavage, and HRP deactivation.
Designed and synthesized high-performance CFT with efficient signal erasure (>95%) while preserving epitope integrity.

Main Results:

Achieved multiplexed protein quantification in single cells with high sensitivity and accuracy.
Demonstrated efficient (>95%) signal removal by mild chemical reagents, preserving target epitopes.
Successfully profiled protein expression heterogeneity and correlations in genetically identical cells and FFPE tissues.

Conclusions:

The developed protein in situ profiling method offers a powerful tool for comprehensive cellular analysis.
The approach allows for accurate quantification of protein expression levels and their relationships in diverse biological samples.
This technique enhances the study of cellular composition, function, and regulation through precise protein profiling.