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Related Concept Videos

Western Blotting01:15

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.
Immunoprecipitation01:20

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 Assay01:33

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 quantified.
Immunogold Electron Microscopy01:20

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 Microscopy01:12

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.
The...

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Related Experiment Video

Updated: Jul 5, 2026

Immunoblot Analysis
16:01

Immunoblot Analysis

Published on: June 20, 2008

Immunoblot detection.

S Gallagher1

  • 1Hoefer Scientific Instruments, San Francisco, California, USA.

Current Protocols in Protein Science
|April 23, 2008
PubMed
Summary
This summary is machine-generated.

Western blotting identifies specific antigens using antibodies. Proteins are separated, transferred to a membrane, and detected with enzyme-linked antibodies and substrates for visualization.

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Immunoblot Analysis
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Published on: June 20, 2008

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Immunoblotting, commonly known as western blotting, is a widely used technique in molecular biology.
  • It enables the specific identification of proteins based on their interaction with antibodies.

Purpose of the Study:

  • To describe the methodology of western blotting for protein detection.
  • To outline the steps involved in identifying and quantifying specific antigens.

Main Methods:

  • Proteins are separated by electrophoresis and transferred to a membrane.
  • Immobilized proteins are probed with primary antibodies specific to the target antigen.
  • Antibody-antigen complexes are detected using secondary antibodies conjugated to enzymes like horseradish peroxidase (HRPO) or alkaline phosphatase (AP).
  • Detection can be direct or utilize an avidin-biotin system.
  • Chromogenic or luminescent substrates are employed for signal visualization.

Main Results:

  • Successful identification and potential quantification of specific antigens.
  • Visualization of antibody-antigen binding through enzymatic reactions.

Conclusions:

  • Western blotting is a versatile and sensitive method for protein analysis.
  • The technique allows for the specific detection and characterization of proteins in complex samples.