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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...

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Related Experiment Video

Updated: Mar 2, 2026

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Published on: September 26, 2020

Antibody Validation by Western Blotting.

Michele Signore¹, Valeria Manganelli², Alex Hodge³

¹Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Via Giano della Bella 34, 00162, Rome, Italy. michele.signore@iss.it.

Methods in Molecular Biology (Clifton, N.J.)

|May 15, 2017

Summary

This summary is machine-generated.

Antibody validation is crucial for reliable biomarker discovery in translational research. Rigorous testing ensures antibody specificity and protein identity, preventing misleading results in clinical studies.

Keywords:

Alkaline phosphatase Antibody Antigen Chemiluminescence Horseradish peroxidase Reverse phase protein microarray SDS-PAGE SNAP i.d.Western blot

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Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

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Published on: January 7, 2019

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Last Updated: Mar 2, 2026

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

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Published on: September 26, 2020

Immunoblot Analysis

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Published on: June 20, 2008

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Published on: January 7, 2019

Area of Science:

Biotechnology
Immunology
Translational Research

Background:

Antibody validation is essential for accurate biomarker discovery in translational research.
Lack of quality control for antibodies can lead to erroneous clinical study results.
Antibody specificity must be confirmed using complex biological samples, not just purified proteins.

Purpose of the Study:

To emphasize the critical importance of antibody validation in translational research.
To detail the process of antibody validation for sensitivity and specificity.
To describe Western blotting techniques for antibody validation.

Main Methods:

Western blotting for protein separation and transfer.
Probing blots to detect specific proteins of interest.
Chemiluminescent detection using manual and vacuum-enhanced systems.

Main Results:

Western blotting demonstrates antibody specificity by detecting a single band at the expected molecular weight in complex samples.
Digital imaging is becoming the standard for immunoblotting detection.
Manual and automated Western blotting systems are available for various applications.

Conclusions:

Thorough antibody validation is critical for the success of translational research and biomarker discovery.
Western blotting is a key technique for confirming antibody specificity.
Accurate antibody validation prevents misleading data in clinical applications.