| Autor: |
Weinberger SR; GenNext Technologies Inc.; sweinberger@gnxtech.com., Chea EE; GenNext Technologies Inc., Sharp JS; GenNext Technologies Inc.; Department of Biomolecular Sciences, School of Pharmacy, University of Mississippi; Department of Chemistry and Biochemistry, University of Mississippi., Misra SK; Department of Biomolecular Sciences, School of Pharmacy, University of Mississippi. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2021 Jun 04 (172). Date of Electronic Publication: 2021 Jun 04. |
| DOI: |
10.3791/61861 |
| Abstrakt: |
Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals ( ▪ OH) to irreversibly label a protein's solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of ▪ OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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