Identifying protein interactions by hydroxyl-radical protein footprinting

Current Protocols in Protein Science
Nick Loizos

Abstract

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.

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Citations

Apr 23, 2008·Current Protocols in Neuroscience·Sean GallagherJohn G R Hurrell
Aug 31, 2012·Molecular Microbiology·George D GlekasGeorge W Ordal

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