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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...
Immunoprecipitation01:20

Immunoprecipitation

Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...

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Related Experiment Video

Updated: Jun 4, 2026

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
07:45

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Published on: June 6, 2022

Optimized protocol to make phospho-specific antibodies that work.

Amy J Archuleta1, Crystal A Stutzke, Kristin M Nixon

  • 1PhosphoSolutions LLC, Aurora, CO, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 4, 2011
PubMed
Summary

This review details a new immunochemical method for creating phospho-specific antibodies. These antibodies precisely track protein activity by recognizing phosphorylated proteins, crucial for understanding cell processes.

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Last Updated: Jun 4, 2026

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

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Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Phosphoproteins regulate critical cellular processes, including cell division and neuronal signaling.
  • Understanding protein activity, not just expression levels, is vital for biological research.
  • Existing methods may not accurately capture dynamic protein phosphorylation states.

Purpose of the Study:

  • To describe the development of a novel immunochemical technique for generating phospho-specific antibodies.
  • To provide a comprehensive guide on producing antibodies that target phosphorylated proteins.
  • To enable researchers to track protein activity with high specificity.

Main Methods:

  • Design and immunization using phosphopeptide immunogens.
  • Affinity purification of generated phospho-specific antibodies.
  • Characterization of antibody phosphospecificity using established methods.

Main Results:

  • Successful development of a technique to produce phospho-specific antibodies.
  • Demonstration of antibodies binding specifically to phosphorylated target proteins.
  • Establishment of protocols for antibody production and validation.

Conclusions:

  • The described methods enable the reliable production of effective phospho-specific antibodies.
  • These antibodies are powerful tools for studying protein activity and cellular signaling.
  • This technique advances the study of phosphoproteins in various biological contexts.