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Enhancing Single-Cell Western Blotting Sensitivity Using Diffusive Analyte Blotting and Antibody Conjugate
Mariia Alibekova Long1,2,3, William K J Benman1,2, Nathan Petrikas1
1Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Analytical Chemistry
|November 17, 2023
Summary
We enhanced single-cell Western blot (scWestern) sensitivity for detecting low-abundance proteins. By transferring blots to nitrocellulose and using enzyme-antibody conjugates, we achieved a 120-fold improvement in detection limits.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Single-cell analysis is crucial for understanding cellular heterogeneity.
- Current protein detection methods for single cells often lack sensitivity and throughput.
- Single-cell Western blots (scWesterns) offer potential but are limited by sensitivity for low-abundance proteins.
Purpose of the Study:
- To improve the sensitivity and limit of detection for single-cell Western blots (scWesterns).
- To enable the detection of low-abundance proteins in single cells.
- To expand the utility of scWesterns by enabling new detection strategies.
Main Methods:
- Decoupled electrophoretic separation from the detection medium by transferring scWesterns to nitrocellulose.
- Utilized enzyme-antibody conjugates for enhanced probing of blotted proteins.
- Compared detection efficiency with traditional in-gel probing methods.
Main Results:
- Achieved a 5.9-fold improvement in the limit of detection by transferring scWesterns to nitrocellulose.
- Further improved the limit of detection to 1000 molecules (a 120-fold increase) using enzyme-antibody conjugates.
- Increased detection of EGFP-expressing cells from 84.5% to 100% compared to in-gel detection.
Conclusions:
- Nitrocellulose-immobilized scWesterns significantly enhance protein detection sensitivity.
- The improved method allows for the detection of low-abundance proteins previously inaccessible.
- This technique expands the range of compatible affinity reagents for single-cell protein analysis.
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