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Related Concept Videos

Phosphorylation01:02

Phosphorylation

51.1K
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...
51.1K
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

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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...
13.4K

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Related Experiment Video

Updated: Sep 10, 2025

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

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Unveiling Phosphorylation Modification Using Phos-tag SDS-PAGE Gel Electrophoresis and In Vitro Kinase Assay.

Qian Zhong1, Yanfang Chen2, Ruiwei Jiang3

  • 1Department of Gastroenterology, Zhongda Hospital, School of Medicine, Southeast University.

Journal of Visualized Experiments : Jove
|August 25, 2025
PubMed
Summary

This study introduces a novel method using Phos-tag gel electrophoresis to detect protein phosphorylation changes in clinical samples without phospho-specific antibodies. This approach enables efficient screening and identification of disease-related phosphorylation sites.

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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
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Last Updated: Sep 10, 2025

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Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Published on: June 30, 2019

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Protein phosphorylation is a critical post-translational modification regulating protein function.
  • Multiple phosphorylation sites on proteins can be modified by various kinases, complicating disease-related studies.
  • Reliance on phospho-specific antibodies limits the analysis of phosphorylation changes, especially when commercial options are unavailable.

Purpose of the Study:

  • To develop and validate a method for detecting and identifying altered protein phosphorylation sites in clinical samples without requiring phospho-specific antibodies.
  • To enable large-scale screening of tissue samples for disease-associated phosphorylation changes.
  • To facilitate the development of specific antibodies for further quantitative and localization analysis.

Main Methods:

  • Utilized Phos-tag gel electrophoresis combined with SDS-PAGE to separate and semi-quantitatively analyze phosphorylated proteins based on altered migration rates.
  • Employed immunoblotting with pan-specific antibodies to identify candidate proteins exhibiting phosphorylation changes.
  • Integrated in vitro kinase assays and mass spectrometry to precisely identify specific phosphorylation sites on target proteins.

Main Results:

  • Demonstrated that Phos-tag gel electrophoresis allows for semi-quantitative analysis of protein phosphorylation changes by detecting slower migration of phosphorylated proteins.
  • Successfully identified target proteins with altered phosphorylation states in disease tissues.
  • Enabled precise identification of phosphorylation sites through in vitro kinase assays and mass spectrometry, bypassing the need for pre-existing phospho-specific antibodies.

Conclusions:

  • The described Phos-tag based method provides a robust, antibody-independent approach for screening and identifying altered phosphorylation in clinical samples.
  • This technique facilitates the discovery of novel disease-related phosphorylation sites, paving the way for targeted therapeutic strategies.
  • The identified sites can be leveraged to generate custom phospho-specific antibodies for detailed functional and clinical investigations.