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Related Concept Videos

Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
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.

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Related Experiment Video

Updated: Jul 5, 2026

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
11:23

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

Published on: June 30, 2019

Detection of phosphorylation by immunological techniques.

B M Sefton1

  • 1The Salk Institute, San Diego, California, USA.

Current Protocols in Protein Science
|April 23, 2008
PubMed
Summary

Detecting protein phosphorylation is crucial. This study details a method using anti-phosphotyrosine antibodies for sensitive detection via immunoblotting with either radiolabeled protein A or enhanced chemiluminescence (ECL).

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Protein phosphorylation is a key post-translational modification regulating cellular processes.
  • Detecting specific phosphorylation events, particularly on tyrosine residues, is essential for understanding signal transduction pathways.
  • Existing methods for detecting protein phosphorylation can be complex or lack sensitivity.

Purpose of the Study:

  • To present a reliable protocol for detecting tyrosine phosphorylation in unlabeled proteins.
  • To provide a method utilizing anti-phosphotyrosine antibodies for sensitive detection.
  • To offer two distinct detection strategies for flexibility and optimization.

Main Methods:

  • Utilizes immunological techniques with specific anti-phosphotyrosine antibodies.

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Oligopeptide Competition Assay for Phosphorylation Site Determination
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Oligopeptide Competition Assay for Phosphorylation Site Determination

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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
11:23

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

Published on: June 30, 2019

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

  • Employs immunoblotting to separate and identify phosphorylated proteins.
  • Describes detection using either radioiodinated (125)I-labeled protein A or enhanced chemiluminescence (ECL).

    • Main Results:

    • Successfully demonstrates the detection of tyrosine phosphorylation using the described protocol.
    • Both (125)I-labeled protein A and ECL provide effective detection of phosphotyrosine.
    • The protocol is applicable to unlabeled protein samples.

    Conclusions:

    • The presented protocol offers a robust and sensitive method for detecting tyrosine phosphorylation.
    • The choice between (125)I-labeled protein A and ECL allows adaptation to different laboratory settings.
    • This technique is valuable for studying signaling pathways involving tyrosine phosphorylation.