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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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Related Experiment Video

Updated: Dec 13, 2025

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Published on: December 11, 2021

Continuous Fc detection for protein A capture process control.

Ujwal Patil¹, Mary Crum², Binh Vu²

¹Biology and Biochemistry, University of Houston, Houston, USA.

Biosensors & Bioelectronics

|July 31, 2020

Summary

This summary is machine-generated.

This study introduces a novel fluorescence-based method for real-time monitoring of monoclonal antibody (mAb) breakthrough during protein A purification. This technology enhances process efficiency by enabling precise detection of antibody loss, preventing underutilization of chromatography columns.

Keywords:

Antibody breakthrough Fluorescence intensity Fluorescence polarization Process analytical technology Protein A chromatography Real-time monitoring

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Last Updated: Dec 13, 2025

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Published on: December 11, 2021

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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

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Published on: December 1, 2018

Area of Science:

Biotechnology
Biopharmaceutical Manufacturing
Analytical Chemistry

Background:

Therapeutic monoclonal antibody (mAb) purification relies on protein A affinity chromatography.
Current methods lack real-time detection of antibody breakthrough in UV-absorbing culture fluid, leading to conservative processing and underutilized capacity.

Purpose of the Study:

To develop and validate a fluorescence-based technology for real-time monitoring of mAb breakthrough during protein A chromatography.
To improve process efficiency and productivity by enabling accurate detection of antibody loss.

Main Methods:

A fluorescence-based monitoring system using fluorescein-labeled Fc-binding ligands was developed.
The system detects shifts in fluorescence polarization and intensity in column effluent.
A flow splitter was used to enable continuous detection at high flow rates.
Python-enabled communication triggered column switching upon detecting breakthrough.

Main Results:

Significant fluorescence intensity shifts were observed in CHO cell culture fluid spiked with human IgG.
The system detected 0.02-0.1 g/L human IgG breakthrough at a flow velocity of 80 cm/h.
A 0.1% breakthrough of a 1 g/L feed triggered column switching.

Conclusions:

The developed fluorescence technology enables rapid, continuous, and reliable monitoring of IgG in process streams.
This method eliminates the need for elaborate sample preparation.
It allows for optimized protein A chromatography, maximizing capacity and productivity.