Autor: |
Chea EE; Department of Pharmaceutical Sciences, University of Maryland Baltimore., Rinas A; AIT Bioscience., Espino JA; Department of Pharmaceutical Sciences, University of Maryland Baltimore., Jones LM; Department of Pharmaceutical Sciences, University of Maryland Baltimore; ljones@rx.umaryland.edu. |
Jazyk: |
angličtina |
Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2020 Mar 11 (157). Date of Electronic Publication: 2020 Mar 11. |
DOI: |
10.3791/60911 |
Abstrakt: |
Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein's solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins. |
Databáze: |
MEDLINE |
Externí odkaz: |
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