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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.

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...

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...

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.

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...

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Related Experiment Video

Updated: Dec 20, 2025

Immunoblot Analysis

Immunoblot Analysis

Published on: June 20, 2008

Immunoblotting.

Larisa Litovchick

Cold Spring Harbor Protocols

|June 3, 2020

Summary

Immunoblotting, commonly known as western blotting, is a key technique for detecting specific proteins in complex samples. This method uses antibodies to identify proteins, determine their levels, and analyze modifications, with broad applications in research and diagnostics.

Area of Science:

Biochemistry
Molecular Biology
Immunology

Background:

Immunoblotting detects specific protein antigens on membranes like nitrocellulose or PVDF.
Protein detection relies on specific antibody binding to the target protein.
Often combined with gel electrophoresis, it's widely known as western blotting.

Purpose of the Study:

To determine the presence and quantity of a protein of interest.
To ascertain the protein's molecular weight and cellular distribution.
To investigate post-translational modifications and utilize epitope tags when specific antibodies are unavailable.

Main Methods:

Immobilizing protein antigens on a membrane support.
Utilizing specific antibodies for immunochemical detection.

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Last Updated: Dec 20, 2025

Immunoblot Analysis

Immunoblot Analysis

Published on: June 20, 2008

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Published on: September 26, 2020

Author Spotlight: Unraveling the Molecular Mechanisms of Brown and Beige Adipocyte Regulation

Author Spotlight: Unraveling the Molecular Mechanisms of Brown and Beige Adipocyte Regulation

Published on: January 5, 2024

Employing gel electrophoresis for protein separation prior to detection.

Main Results:

Enables quantification of protein levels and determination of molecular weight.
Facilitates study of protein modifications like phosphorylation and acetylation.
Allows detection using epitope tags (e.g., HA, Flag, c-Myc, GST) when primary antibodies are absent.

Conclusions:

Immunoblotting is versatile for protein analysis, including modifications and expression levels.
It serves critical roles in research, clinical diagnostics, and forensic medicine.
Essential for antibody characterization from various biological samples.