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.
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© 2004 Humana Press Inc., Totowa, NJ
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Loizos, N. (2004). Mapping Protein-Ligand Interactions by Hydroxyl-Radical Protein Footprinting. In: Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 261. Humana Press. https://doi.org/10.1385/1-59259-762-9:199
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DOI: https://doi.org/10.1385/1-59259-762-9:199
Publisher Name: Humana Press
Print ISBN: 978-1-58829-120-2
Online ISBN: 978-1-59259-762-8
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