Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Akira Kitamura; Ai Fujimoto; Masataka Kinjo

doi:10.3791/62576

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

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Biology
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JoVE Journal Biology

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

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14:04 min

April 25, 2021

DOI: 10.3791/62576-v

Akira Kitamura¹, Ai Fujimoto¹, Masataka Kinjo¹

¹Laboratory for Molecular Cell Dynamics, Faculty of Advanced Life Science,Hokkaido University

Overview

This study presents a procedure utilizing fluorescence correlation spectroscopy (FCS) to measure protein oligomers and aggregation in cell lysates and live cells. The focus is on analyzing protein aggregates associated with amyotrophic lateral sclerosis, highlighting the significance of FCS in delineating molecular interactions within biological samples.

Key Study Components

Research Area

Protein aggregation analysis
Neurodegenerative disorder research
Molecular interactions

Background

Fluorescence correlation spectroscopy (FCS) is a sensitive technique developed in the 1970s, improved with confocal microscopy in the 1990s.
FCS is vital for understanding molecular sizes and diffusion properties.
Protein aggregation is critical for studying neurodegenerative diseases.

Methods Used

Fluorescence correlation spectroscopy
Cell lysates from Neuro2a cells
Confocal microscopy systems for real-time analysis

Main Results

Successful measurement of protein aggregates in live cells.
Diffusion properties provided insights into molecular interactions.
Spikes in FCS data indicated soluble oligomers or aggregates.

Conclusions

The method effectively demonstrates protein aggregation dynamics in living systems.
This research contributes to understanding neurodegenerative disorders at the molecular level.

Frequently Asked Questions

What is fluorescence correlation spectroscopy (FCS)?

FCS is a technique used to analyze the dynamics of molecules in solution and within live cells by measuring fluctuations in fluorescence intensity.

How does FCS help in studying protein aggregation?

FCS can detect and quantify the movement and interactions of protein aggregates, providing insights into their formation and implications in diseases.

What differentiates live cell measurements from lysate measurements in FCS?

Live cell measurements provide real-time data on molecular interactions, while lysate measurements offer bulk analysis of cellular components.

Why is studying protein aggregates important for neurodegenerative disorders?

Protein aggregates are associated with the pathology of many neurodegenerative diseases, making their analysis crucial for understanding disease mechanisms.

What kind of cells were used in this study?

Neuro2a cells, a neuronal cell line, were used for experiments to study protein aggregation related to motor neuron diseases.

What role does transfection play in the experiments?

Transfection enables the expression of specific proteins tagged with fluorescent markers, facilitating real-time monitoring via FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

The best principle of the Fluorescence correlation spectroscopy, FCS, was invented initially in the 1970s. In the 1990s, important and many improvements for FCS was established by combining with a confocal apparatus microscopy. Since then, FCS has been used for many chemical and virus cure applications, such as molecular interactions and protein aggregation analysis.

Protein aggregation is a hallmark of neurodegenerative disorders. Fluorescence brightness are single particles in action of molecular sizes explained by diffusion properties. It's very important in measuring protein aggregations because it can determine projected life cycles of molecules, but also distinguish the homo or hetero polymerization.

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