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Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Published on: May 17, 2010

Ultrasensitive and Multiplexed Protein Imaging with Clickable and Cleavable Fluorophores.

Thai Pham¹, Yi Chen¹, Joshua Labaer¹

¹Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, United States.

Analytical Chemistry

|April 25, 2024

Summary

Researchers developed a new method for highly sensitive, multiplexed in situ protein profiling using readily available antibodies. This technique enables detailed cell analysis in tissues, advancing spatial biology and understanding cellular interactions.

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Area of Science:

Biotechnology
Molecular Biology
Cell Biology

Background:

Single-cell spatial proteomic analysis is crucial for understanding complex biological systems.
Current multiplexed protein imaging methods face limitations in sensitivity, multiplexing capacity, and technical complexity.

Purpose of the Study:

To develop a highly sensitive and multiplexed in situ protein profiling method.
To overcome the limitations of existing protein imaging technologies.

Main Methods:

Utilized off-the-shelf antibodies conjugated with horseradish peroxidase (HRP).
Employed cleavable fluorophores and click chemistry for sequential staining and imaging.
Implemented repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage.

Main Results:

Successfully quantified 28 different proteins in single cells within human FFPE tonsil tissue.
Achieved the highest multiplexing capacity reported for tyramide signal amplification (TSA) methods.
Classified approximately 820,000 cells into distinct clusters based on protein expression and microenvironment.
Revealed specific cell-cell interactions and regional compositions within the tissue.

Conclusions:

The developed method offers a highly sensitive and multiplexed approach for in situ protein profiling.
This technique significantly advances the field of spatial biology by enabling detailed analysis of cellular composition, organization, and interaction.
The findings provide a deeper understanding of tissue architecture and cell-cell communication.