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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.

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Related Experiment Video

Updated: Jun 27, 2026

Immunoblot Analysis
16:01

Immunoblot Analysis

Published on: June 20, 2008

Immunoblot analysis.

Sean Gallagher1, Deb Chakavarti

  • 1UVP, LLC, USA.

Journal of Visualized Experiments : Jove
|December 11, 2008
PubMed
Summary
This summary is machine-generated.

Immunoblotting, or western blotting, is a sensitive protein detection method using antigen-antibody recognition. This technique enables precise protein characterization and identification through gel electrophoresis and membrane transfer.

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Immunoblot Analysis
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Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Immunoblotting (western blotting) is a key technique for protein detection and characterization.
  • It relies on the principle of specific antigen-antibody interactions.
  • The assay is known for its speed and sensitivity.

Purpose of the Study:

  • To provide detailed protocols for immunoblotting.
  • To outline methods for protein separation, transfer, and detection.
  • To cover various visualization techniques.

Main Methods:

  • Protein solubilization and separation via gel electrophoresis.
  • Quantitative transfer of separated proteins to a solid support membrane (nitrocellulose, PVDF, or nylon).
  • Immunoprobing with specific antibodies, followed by detection using chromogenic or chemiluminescent substrates.

Main Results:

  • Demonstrates the high sensitivity of immunoblotting, capable of detecting as little as 1 ng of antigen.
  • Highlights the utility of the technique in identifying specific antigens.
  • Confirms the effectiveness of both polyclonal and monoclonal antibodies in antigen recognition.

Conclusions:

  • Immunoblotting is a versatile and sensitive method for protein analysis.
  • The provided protocols facilitate accurate protein separation, blotting, immunoprobing, and visualization.
  • This technique is crucial for characterizing proteins in various biological contexts.