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Related Experiment Video

Updated: Jan 10, 2026

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

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Published on: July 12, 2014

Identification of Previously Unknown DNA-Binding Proteins Using DNA Affinity/Pull-Down Methods Followed by Mass

Brandon L Jutras¹, Kelly Babb¹, Nerina Jusufovic¹

¹Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky.

Current Protocols

|November 25, 2025

Summary

This study details a method to isolate and identify novel DNA-binding proteins from bacteria. The technique uses DNA affinity chromatography and mass spectrometry for protein identification, applicable to various organisms and RNA-binding proteins.

Keywords:

DNA‐affinity DNA‐binding protein identify unknown protein mass spectrometry pull‐down

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Last Updated: Jan 10, 2026

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

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Published on: July 12, 2014

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

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Published on: April 1, 2017

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Published on: February 25, 2012

Area of Science:

Molecular Biology
Proteomics
Biochemistry

Background:

Identifying DNA-binding proteins is crucial for understanding gene regulation.
Existing methods may have limitations in specificity or scope.

Purpose of the Study:

To present a robust protocol for isolating and identifying novel nucleic acid-binding proteins.
To adapt methods for bacterial DNA-binding proteins, with potential for eukaryotes, archaea, and RNA-binding proteins.

Main Methods:

DNA sequence immobilization on beads.
Incubation with bacterial cytoplasmic extract.
Differential washing based on salt concentration to elute specific DNA-binding proteins.
Identification of eluted proteins using mass spectrometry.

Main Results:

Successful isolation of specific DNA-binding proteins from bacterial extracts.
Demonstration of the protocol's adaptability for different organisms and nucleic acid types.
Identification of eluted proteins via proteomic analysis.

Conclusions:

The presented DNA affinity chromatography protocol is effective for isolating specific DNA-binding proteins.
The method is versatile and can be applied to identify nucleic acid-binding proteins across different domains of life and for RNA targets.