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Western Blotting

Western Blotting

Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.

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Related Experiment Video

Updated: Apr 28, 2026

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Published on: August 22, 2018

Single-cell western blotting.

Alex J Hughes¹, Dawn P Spelke², Zhuchen Xu³

¹1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] [4].

|June 2, 2014

Summary

This summary is machine-generated.

We developed single-cell western blots (scWesterns) to measure protein variation between cells. This method analyzes protein expression in thousands of individual cells rapidly, offering high sensitivity and multiplexing capabilities for complex biological studies.

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Western Blotting: Sample Preparation to Detection

Western Blotting: Sample Preparation to Detection

Published on: October 14, 2010

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Published on: April 9, 2019

Related Experiment Videos

Last Updated: Apr 28, 2026

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Published on: August 22, 2018

Western Blotting: Sample Preparation to Detection

Western Blotting: Sample Preparation to Detection

Published on: October 14, 2010

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Published on: April 9, 2019

Area of Science:

Biochemistry
Cell Biology
Proteomics

Background:

Understanding cell-to-cell variability in protein function is crucial for biological research.
Existing single-cell protein analysis methods face limitations in sensitivity and antibody fidelity.
High-throughput, high-resolution protein analysis is needed to study complex cellular processes.

Purpose of the Study:

To develop a novel method for high-throughput single-cell western blotting (scWestern).
To enable precise measurement of cell-to-cell variation in protein-mediated functions.
To overcome current limitations in single-cell protein quantification.

Main Methods:

Development of a photoactive polyacrylamide gel on a microscope slide for in situ western blotting.
Integration of microwells for single-cell capture, lysis, electrophoresis, and photoinitiated protein immobilization.
Antibody probing for multiplexed protein target detection at the single-cell level.

Main Results:

Concurrent execution of approximately 10^3 single-cell western blots (scWesterns) within 4 hours.
Quantification of target proteins with detection thresholds below 30,000 molecules per cell.
Successful multiplexing of up to 11 protein targets per single cell.
Demonstrated application in monitoring neural stem cell differentiation and mitogen response.
Integration with Fluorescence-Activated Cell Sorting (FACS) for analysis of small cell populations.

Conclusions:

The scWestern method provides a versatile and powerful tool for single-cell protein analysis.
This technique overcomes key limitations in antibody fidelity and sensitivity compared to other methods.
scWestern enables high-resolution studies of protein expression in complex cell populations.
The method facilitates deeper understanding of cell differentiation and cellular responses.