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Related Experiment Video

Updated: Jun 28, 2026

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies
10:01

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Published on: November 28, 2017

RNA-protein crosslink mapping using TEV protease.

Ian A Turner1, Chris M Norman, Mark J Churcher

  • 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, United Kingdom.

Methods in Molecular Biology (Clifton, N.J.)
|November 5, 2008
PubMed
Summary

This study introduces a new method for mapping RNA-protein binding sites using a peptide tag and TEV protease. This approach overcomes limitations of traditional methods for identifying crosslink sites in proteins.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Proteomics

Background:

  • Characterizing RNA-protein interactions is crucial for understanding cellular functions.
  • Traditional methods for mapping RNA-protein crosslinks can be ambiguous, especially for large proteins or multiple binding sites.

Purpose of the Study:

  • To develop an improved method for unambiguous physical mapping of RNA-protein crosslinks.
  • To overcome the limitations of traditional protein fragmentation techniques.

Main Methods:

  • Insertion of a peptide tag (ENLYFQG) recognized by TEV protease into proteins.
  • TEV protease digestion of crosslinked proteins.
  • Fractionation of digestion products using SDS-PAGE.
  • Visualization of labeled fragments via phosphorimaging.

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Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
11:33

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Published on: December 17, 2013

Related Experiment Videos

Last Updated: Jun 28, 2026

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies
10:01

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Published on: November 28, 2017

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
11:33

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Published on: December 17, 2013

Main Results:

  • The novel method allows for precise physical mapping of RNA-protein crosslinks.
  • This technique provides unambiguous identification of crosslinked sites, even in complex scenarios.

Conclusions:

  • The developed method offers a robust alternative for mapping RNA-protein interactions.
  • This technique enhances the characterization of RNA-binding proteins and their functional sites.