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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: Dec 28, 2025

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Published on: January 25, 2020

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy.

Avani V Pisapati¹, Yi Wang², Megan E Blauch³

¹Department of Bioengineering, Lehigh University.

Journal of Visualized Experiments : Jove

|February 18, 2020

Summary

This summary is machine-generated.

This study presents a fluorescence microscopy protocol to visualize single biomolecules

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Last Updated: Dec 28, 2025

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Published on: January 25, 2020

Ensemble Force Spectroscopy by Shear Forces

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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Published on: May 29, 2011

Area of Science:

Biophysics
Fluid Mechanics
Polymer Science

Background:

Single-molecule behavior under mechanical forces is crucial for biological processes.
Existing methods like AFM and FRET have limitations in temporal resolution or inferential capabilities.
Fluorescence microscopy offers real-time, in situ visualization of single molecules.

Purpose of the Study:

To develop and demonstrate a protocol for capturing single biomolecule conformational changes under shear flow.
To enable real-time observation of molecular behavior in controlled flow environments.

Main Methods:

Utilizing microfluidic channels to generate controlled shear flow via a syringe pump.
Immobilizing labeled biomolecules (von Willebrand factor and lambda DNA) on channel surfaces.
Employing total internal reflection (TIRF) and confocal fluorescence microscopy for continuous monitoring.

Main Results:

Successfully visualized reversible unraveling dynamics of von Willebrand factor (VWF).
Observed conformational changes in lambda DNA under varying shear flow conditions.
Demonstrated the protocol's ability to capture dynamic molecular responses to flow.

Conclusions:

The developed protocol provides a powerful tool for studying single-molecule dynamics in flow.
Insights into VWF regulation in blood and macromolecular biophysics from DNA conformation studies.
Broad applicability to polymers, biopolymers, and complex fluid rheology.