Mapping Protein-Ligand Interactions by Hydroxyl-Radical Protein Footprinting

  • Nick Loizos
Part of the Methods in Molecular Biology book series (MIMB, volume 261)

Abstract

Hydroxyl-radical protein footprinting is a 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 show regions of the protein that change their susceptibility to cleavage while bound to a ligand.

Key Words

Hydroxyl-radical protein footprinting macromolecular interactions protein end-labeling Fe-EDTA 

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Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Nick Loizos
    • 1
  1. 1.Department of Protein ChemistryImClone Systems Inc.New York

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