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Related Experiment Video

Updated: Nov 5, 2025

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Published on: July 6, 2014

Identifying Protein-(Hydroxy)Methylated DNA Interactions Using Quantitative Interaction Proteomics.

Velin Marita Sequeira¹, Michiel Vermeulen²

¹Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Oncode Institute, Radboud University Nijmegen, Nijmegen, The Netherlands.

Methods in Molecular Biology (Clifton, N.J.)

|May 19, 2021

Summary

This summary is machine-generated.

This study details a method for mapping protein-DNA interactions using DNA affinity purification and mass spectrometry. It identifies proteins binding to specific DNA sequences, including methylated DNA.

Keywords:

Dimethyl labeling Mass spectrometry Methylation readers Protein–methylated DNA interactions Quantitative proteomics

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Area of Science:

Molecular Biology
Proteomics
Genomics

Background:

Comprehensive mapping of protein-DNA interactions is crucial for understanding cellular processes.
Quantitative mass spectrometry-based proteomics coupled with DNA affinity purification offers an unbiased approach.
Identifying specific DNA-binding proteins is essential for deciphering gene regulation and epigenetic mechanisms.

Purpose of the Study:

To describe a detailed protocol for mapping protein-DNA interactions.
To identify specific interaction partners for (hydroxy)methylated and non-methylated DNA sequences.
To combine DNA affinity purification with chemical stable isotope labeling for quantitative analysis.

Main Methods:

DNA affinity purification from crude nuclear extracts.
Quantitative mass spectrometry-based proteomics.
Chemical stable isotope labeling using dimethyl labeling.

Main Results:

The protocol enables the identification of proteins that specifically bind to defined DNA sequences.
The method allows for the differentiation of binding partners for methylated versus non-methylated DNA.
Quantitative analysis provides insights into the relative abundance of interacting proteins.

Conclusions:

This workflow provides a robust method for comprehensive mapping of protein-DNA interactions.
The protocol is applicable to various cell or tissue types of interest.
The approach facilitates the study of epigenetic modifications and their associated protein partners.