UNIT 19.9 Identifying Protein Interactions by Hydroxyl-Radical Protein Footprinting

  1. Nick Loizos

Published Online: 1 NOV 2004

DOI: 10.1002/0471140864.ps1909s38

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Loizos, N. 2004. Identifying Protein Interactions by Hydroxyl-Radical Protein Footprinting. Current Protocols in Protein Science. 38:19.9:19.9.1–19.9.11.

Author Information

  1. ImClone Systems Incorporated, New York, New York

Publication History

  1. Published Online: 1 NOV 2004
  2. Published Print: OCT 2004


Hydroxyl-radical protein footprinting is a straightforward and direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end-label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that shows regions of the protein that change their susceptibility to cleavage while bound to a ligand.


  • hydroxyl-radical;
  • protein footprinting;
  • macromolecular interactions;
  • protein end-labeling;
  • Fe-EDTA