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Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
Renjie Liao1, Thai Pham1, Diego Mastroeni2,3
1Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA.
Cells
|April 5, 2020
Summary
This study introduces a novel in-situ protein analysis method using cleavable biotin-conjugated antibodies and fluorescent streptavidin (CFS). This technique significantly enhances protein detection sensitivity for multiplexed profiling in individual cells and FFPE tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Highly sensitive and multiplexed in-situ protein analysis is vital for understanding physiology and disease.
- Current in-situ proteomics methods have limitations in sensitivity and multiplexing capabilities.
Purpose of the Study:
- To develop an advanced approach for highly sensitive and multiplexed in-situ protein analysis.
- To enhance protein detection sensitivity beyond current in-situ proteomics methods.
Main Methods:
- Utilized cleavable biotin-conjugated antibodies for target recognition.
- Employed cleavable fluorescent streptavidin (CFS) for protein staining.
- Implemented layer-by-layer signal amplification with orthogonal antibodies and CFS.
- Developed a chemical cleavage and blocking strategy for iterative analysis cycles.
Main Results:
- Achieved at least a 10-fold increase in protein detection sensitivity compared to existing methods.
- Demonstrated unambiguous detection and profiling of multiple proteins in the same cells.
- Successfully applied the method for protein quantification in formalin-fixed paraffin-embedded (FFPE) tissues.
Conclusions:
- The developed method enables highly sensitive, multiplexed in-situ protein profiling at the single-cell level.
- This approach overcomes limitations of current techniques and is applicable to FFPE tissues.
- Facilitates deeper understanding of cellular processes and disease pathogenesis through comprehensive protein analysis.
