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Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
Published on: April 25, 2021
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Fluorogenic detection of protein aggregates in live cells using the AggTag method
1Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Methods in Enzymology
|June 2, 2020
Summary
Researchers developed AggTag, a novel fluorescence microscopy method to detect protein aggregation in live cells. This technique visualizes previously undetectable soluble oligomers and enables simultaneous tracking of multiple protein interactions.
Area of Science:
- Biochemistry
- Cell Biology
- Microscopy
Background:
- Protein aggregation, involving self-assembly of misfolded proteins, is implicated in neurodegenerative diseases.
- Current methods struggle to detect and study protein aggregation species within live cells.
- There is a critical need for advanced imaging techniques to investigate protein aggregation dynamics.
Purpose of the Study:
- To introduce AggTag, a new fluorescence microscopy method for detecting protein aggregation in live cells.
- To enable visualization of soluble misfolded oligomers, which are difficult to detect with existing techniques.
- To provide a detailed protocol for researchers studying protein aggregation in mammalian cell lines.
Main Methods:
- Developed AggTag, a genetic fusion system linking a protein of interest (POI) to a protein tag.
- Utilized an AggTag probe with a fluorophore that activates upon binding to protein aggregates.
- Implemented a dual-color, orthogonal tagging system for simultaneous detection of co-aggregation between two POIs.
Main Results:
- AggTag successfully reports on protein aggregation in live cells using fluorescence microscopy.
- The method enables the detection of soluble misfolded oligomers, overcoming limitations of conventional approaches.
- Demonstrated simultaneous detection of co-aggregation between two different POIs.
Conclusions:
- AggTag offers a powerful new tool for studying protein aggregation in live cells.
- This method advances the ability to investigate the role of protein aggregation in cellular processes and diseases.
- The provided procedures facilitate the application of AggTag in diverse research settings.
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