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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.

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Updated: Jul 5, 2026

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Published on: June 30, 2019

Antiphosphotyrosine blotting.

Jeffrey N Siegel¹

¹Naval Medical Research Institute, Bethesda, Maryland.

Current Protocols in Immunology

|April 25, 2008

Summary

This summary is machine-generated.

Antiphosphotyrosine blotting detects tyrosine-phosphorylated proteins using specific antibodies. This method offers sensitive detection of these substrates and other proteins via immunoblotting and colorimetric detection.

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Area of Science:

Biochemistry
Molecular Biology
Immunology

Background:

Tyrosine phosphorylation is a critical post-translational modification regulating numerous cellular processes.
Detecting tyrosine-phosphorylated proteins is essential for understanding cell signaling pathways.
Existing methods may lack sensitivity or require radioactive detection.

Purpose of the Study:

To describe a sensitive and robust protocol for antiphosphotyrosine blotting.
To detail the steps for cell lysis, immunoprecipitation, and detection of tyrosine-phosphorylated proteins.
To compare different detection systems for optimal results.

Main Methods:

Cell lysis and protein extraction.
Immunoprecipitation using antiphosphotyrosine antibodies.
Electrophoretic separation (SDS-PAGE) and immunoblotting.
Colorimetric detection using alkaline phosphatase or radiolabeled detection systems.

Main Results:

The described protocol provides highly sensitive detection of tyrosine-phosphorylated substrates.
The method is adaptable for detecting various proteins transferred to nitrocellulose membranes.
Both alkaline phosphatase and (125)I-labeled Staphylococcus protein A systems yield effective results.

Conclusions:

Antiphosphotyrosine blotting is a valuable technique for identifying tyrosine-phosphorylated proteins.
The protocol offers high sensitivity and versatility for protein detection.
The alkaline phosphatase system provides a non-radioactive alternative with excellent resolution and sensitivity.