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Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative
Rina Kunishige1,2, Masayuki Murata1,2,3, Fumi Kano1,2
1Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan.
Frontiers in Cell and Developmental Biology
|January 5, 2023
Summary
A new cell resealing technique using streptolysin O (SLO) enhances Trim-Away protein degradation. This method simplifies antibody delivery for studying protein function and cellular effects.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- The Trim-Away technology facilitates rapid degradation of endogenous proteins.
- Current antibody delivery methods for Trim-Away are limited, requiring specialized equipment or skills.
- There is a need for convenient, scalable antibody delivery methods for adherent cells.
Purpose of the Study:
- To develop an optimized cell resealing technique for Trim-Away protein degradation.
- To enable convenient and scalable delivery of antibodies into cells for Trim-Away.
- To couple Trim-Away with advanced imaging analysis for quantitative cellular studies.
Main Methods:
- Developed a cell resealing technique using streptolysin O (SLO) for antibody delivery.
- Applied the technique to HEK293T, HeLa, and HK-2 cell lines.
- Utilized cyclic immunofluorescence and microscopic image-based analysis for single-cell multiplexed imaging.
Main Results:
- Successfully demonstrated Trim-Away protein degradation of IKKα and mTOR using the SLO-mediated resealing method.
- Validated the system for antibody screening in Trim-Away applications.
- Achieved compensation for signal-to-noise variations through quantitative image analysis.
Conclusions:
- The developed cell resealing technique offers a convenient and scalable method for Trim-Away protein degradation.
- Coupling Trim-Away with microscopic image analysis enables precise elucidation of protein function and degradation effects.
- This integrated approach enhances the quantitative and precise analysis of cellular functions affected by protein degradation.
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