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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.

Southern Blot

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...

Labeling DNA Probes

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...

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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Updated: May 1, 2026

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

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Western blotting using chemiluminescent substrates.

Alice Alegria-Schaffer¹

¹Thermo Fisher Scientific, Rockford, IL, USA.

Methods in Enzymology

|March 29, 2014

Summary

Western blotting is a widely used technique for identifying and quantifying specific proteins within complex biological samples. This method allows for the indirect detection of proteins after they have been immobilized onto a membrane.

Keywords:

Horseradish peroxidase (HRP)Nitrocellulose membrane Polyvinylidene fluoride (PVDF) membrane Protein transfer SDS-PAGE Western blotting

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Western Blotting: Sample Preparation to Detection

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Last Updated: May 1, 2026

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Western Blotting: Sample Preparation to Detection

Western Blotting: Sample Preparation to Detection

Published on: October 14, 2010

Area of Science:

Biochemistry
Molecular Biology
Immunology

Background:

Western blotting is a cornerstone technique in molecular biology.
It is essential for protein analysis in various research fields.

Purpose of the Study:

To describe the fundamental principles of Western blotting.
To highlight its utility in protein identification and quantification.

Main Methods:

Protein samples are separated, typically by gel electrophoresis.
Separated proteins are transferred to a solid support membrane (nitrocellulose or PVDF).
Specific proteins are detected using antibodies.

Main Results:

Successful identification of target proteins from complex mixtures.
Quantification of protein levels is achievable.

Conclusions:

Western blotting remains a vital tool for protein research.
The technique's reliability supports its widespread application.