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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.

Enzyme-Linked Immunosorbent Assay

Enzyme-Linked Immunosorbent Assay

In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or...

Immunoprecipitation

Immunoprecipitation

Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...

Immunocytochemistry and Immunohistochemistry

Immunocytochemistry and Immunohistochemistry

Immunocytochemistry (ICC) and immunohistochemistry (IHC) are techniques that use antibodies to check for specific proteins or antigens in a sample. The technique was first published by Albert Coons in 1941 to detect the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus. Immunocytochemistry helps localization of proteins or antigens in individual cells like blood cells, stem cells, etc., while immunohistochemistry does the same for tissue samples.
These...

Immunogold Electron Microscopy

Immunogold Electron Microscopy

Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.

Immunofluorescence Microscopy

Immunofluorescence Microscopy

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.

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Related Experiment Video

Updated: Mar 23, 2026

Immunoblot Analysis

Immunoblot Analysis

Published on: June 20, 2008

Immunoblotting and Immunodetection.

Duojiao Ni¹, Peng Xu², Sean Gallagher¹

¹UVP, LLC, Upland, California.

Current Protocols in Molecular Biology

|April 3, 2016

Summary

This summary is machine-generated.

Western blotting is a key technique for identifying specific antigens using antibodies. This protocol details protein solubilization, separation, membrane transfer, and immunodetection for antigen analysis.

Keywords:

alkaline phosphatase antibodies horseradish peroxidase immunoblot western blot

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Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Published on: September 26, 2020

Area of Science:

Biochemistry
Molecular Biology
Immunology

Background:

Immunoblotting, commonly known as western blotting, is a widely used technique in molecular biology and immunology.
It enables the detection and identification of specific proteins (antigens) within a complex mixture.
The process relies on the high specificity of antibody-antigen interactions.

Purpose of the Study:

To provide a comprehensive protocol for performing immunoblotting (western blotting).
To outline the essential steps from sample preparation to final detection.
To ensure reproducibility and clarity for researchers utilizing this technique.

Main Methods:

Protein sample solubilization using sodium dodecyl sulfate (SDS) and reducing agents.
Separation of proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Electrophoretic transfer of separated proteins onto a membrane, monitored by Ponceau S staining.
Immunodetection involving blocking nonspecific sites, probing with primary and secondary antibodies, and signal detection via fluorescence or chromogenic/luminescent substrates.

Main Results:

Successful identification of specific antigens through antibody recognition.
Demonstration of the sequential steps involved in western blotting, from sample preparation to detection.
Validation of membrane stripping and reprobing for multiple antigen detection.

Conclusions:

Western blotting is a robust and versatile method for specific antigen detection.
The detailed protocol ensures efficient and reliable execution of the technique.
This method facilitates the analysis of protein expression and interactions in various biological contexts.