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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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The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular fractionation. These isolates are used to study specific cellular components, analyze localized protein activity, and are even employed in diagnostics. Fractionation is typically achieved using centrifugation methods, the most common being density-gradient and differential centrifugation.
Differential Centrifugation
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Related Experiment Video

Updated: Sep 11, 2025

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Published on: April 6, 2022

Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution.

Lorena Alamillo¹, Alexander Black¹, Maggie P Y Lam^1,2,3

¹Deptartment of Medicine, University of Colorado School of Medicine, Aurora, CO, USA.

|August 13, 2025

Summary

This summary is machine-generated.

This study introduces Simultaneous Proteome Localization and Turnover (SPLAT) analysis, a novel method to measure protein turnover rates and subcellular localization across the entire proteome. SPLAT provides a comprehensive view of protein dynamics within cellular compartments, crucial for understanding age-associated diseases.

Keywords:

Mass spectrometry Proteomics Spatial proteomics Subcellular localization Temporal proteomics Turnover

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Last Updated: Sep 11, 2025

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Published on: April 6, 2022

Probing Structural and Dynamic Properties of Trafficking Subcellular Nanostructures by Spatiotemporal Fluctuation Spectroscopy

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Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Published on: February 4, 2018

Area of Science:

Cell Biology
Proteomics
Biochemistry

Background:

Protein homeostasis, involving synthesis and degradation, is vital for cellular function and implicated in aging.
Existing methods for studying protein turnover in subcellular compartments are often invasive or limited in scope.
Understanding protein dynamics within specific cellular locations is essential for disease research.

Purpose of the Study:

To develop a novel protocol for simultaneous measurement of proteome-wide protein turnover rates and subcellular localization.
To provide an unbiased approach for assessing compartment-specific protein dynamics.
To enable detailed analysis of protein spatial and temporal distributions.

Main Methods:

Simultaneous Proteome Localization and Turnover (SPLAT) analysis.
Dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) for temporal resolution.
Multi-step differential ultracentrifugation for subcellular localization.
2D liquid chromatography fractionation and tandem mass tags (TMT) for enhanced depth and reduced acquisition time.

Main Results:

SPLAT analysis successfully measures protein turnover rates and subcellular localization simultaneously.
The protocol provides proteome-wide, compartment-specific turnover rates.
It reveals both spatial and temporal distributions of proteins, including temporally distinct localizations within protein pools.
Hyperplexing of dynamic SILAC and TMT LOPIT-DC enhances data acquisition and depth.

Conclusions:

SPLAT analysis offers a powerful, unbiased tool for studying protein dynamics across subcellular compartments.
This method significantly advances the ability to compare organellar proteome turnover rates.
The protocol facilitates a deeper understanding of protein homeostasis and its role in age-associated diseases.