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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: Apr 27, 2026

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Published on: August 17, 2015

Determining absolute protein numbers by quantitative fluorescence microscopy.

Jolien Suzanne Verdaasdonk¹, Josh Lawrimore¹, Kerry Bloom¹

¹Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Methods in Cell Biology

|June 30, 2014

Summary

This summary is machine-generated.

Quantitative microscopy methods like fluorescence correlation spectroscopy and ratiometric analysis can determine absolute protein numbers. This counting technique enhances understanding of protein complex composition and cellular dynamics.

Keywords:

Counting FCS Fluorescence Fluorescence standards Photobleaching Quantitative imaging Ratiometric

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Area of Science:

Cellular Biology
Biophysics
Quantitative Biology

Background:

Biological questions increasingly utilize quantitative analytical tools for protein complex analysis.
Understanding absolute protein numbers is crucial for insights into cellular organization, function, and mathematical modeling.

Purpose of the Study:

To outline and describe microscopy-based methods for determining absolute protein numbers.
To provide a detailed procedure for ratiometric comparison analysis.

Main Methods:

Fluorescence correlation spectroscopy
Stepwise photobleaching
Ratiometric comparison of fluorescence intensity to known standards

Main Results:

Discussion of fluorescently labeled proteins used as standards for quantification.
Detailed procedure for absolute protein number determination via ratiometric comparison.

Conclusions:

Quantitative microscopy offers a simple yet powerful tool for analyzing protein complex composition.
Accurate protein counting advances our understanding of cellular dynamics and organization.