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Related Concept Videos

Western Blotting01:15

Western Blotting

21.7K
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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Enzyme-Linked Immunosorbent Assay01:33

Enzyme-Linked Immunosorbent Assay

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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...
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Immunocytochemistry and Immunohistochemistry01:22

Immunocytochemistry and Immunohistochemistry

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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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Immunogold Electron Microscopy01:20

Immunogold Electron Microscopy

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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.
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Immunoprecipitation01:20

Immunoprecipitation

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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...
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Immunofluorescence Microscopy01:12

Immunofluorescence Microscopy

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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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Updated: Mar 17, 2026

Immunoblot Analysis
16:01

Immunoblot Analysis

Published on: June 20, 2008

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Immunoblotting and Immunodetection.

Duojiao Ni1, Peng Xu2, Sean Gallagher1

  • 1UVP, LLC, Upland, California.

Current Protocols in Immunology
|August 2, 2016
PubMed
Summary
This summary is machine-generated.

Western blotting is a key technique for detecting specific proteins using antibodies. This protocol details sample preparation, separation, transfer, and immunodetection for reliable antigen identification.

Keywords:
alkaline phosphataseantibodieshorseradish peroxidaseimmunoblotwestern blot

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Immunoblotting, commonly known as western blotting, is a fundamental technique in molecular biology.
  • It is essential for the specific detection of target proteins using antibodies.

Purpose of the Study:

  • To provide a comprehensive protocol for performing immunoblotting.
  • To cover all steps from protein sample preparation to final detection and reprobing.

Main Methods:

  • Protein sample solubilization using SDS and reducing agents.
  • Separation of proteins via SDS-PAGE.
  • Electrophoretic transfer of proteins to a membrane.
  • Immunodetection using primary and secondary antibodies.
  • Detection via fluorescent, chromogenic, or luminescent substrates.

Main Results:

  • Successful identification of specific antigens.
  • Demonstration of the complete western blotting workflow.
  • Capability for membrane stripping and reprobing for sequential analysis.

Conclusions:

  • The provided protocols enable robust and versatile protein analysis using western blotting.
  • This method is crucial for identifying specific antigens in complex biological samples.
  • The technique allows for sensitive and specific detection of target proteins.