The dynamic and unstable nature of protein nitrosothiols (PSNOs) derived from complex biological matrices (like cell lysates) make them unsuitable for proteomic/biochemical analysis in vitro. In an attempt to increase the stability of cell-derived PSNOs, scientists have devised methods to derivatize thiols undergoing nitrosylation, with a suitable molecule, to yield a stable adduct that could easily be detected using appropriate antibodies. The Biotin Switch Assay (BTSA) is currently the most widely used method for tagging PSNOs; however, the error-prone and cumbersome nature of the BTSA protocol prompted the development of alternative mechanisms of tagging cell-derived PSNOs. One such method is the immuno-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which effectively overcomes the shortcomings of the BTSA and proves to be a promising alternative. Here we describe the protocol for DMPO-based PSNO labeling and subsequent proteomic analysis by western blotting with an anti-DMPO antibody. © 2021 Wiley Periodicals LLC. Basic Protocol: Labeling of cell-derived PSNOs by DMPO-based immuno-spin trapping and their subsequent analysis by immunostaining.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1002/cpz1.262 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
A reciprocal relationship between mitochondria and lipid peroxidation determines the chondrocyte intracellular redox environment.
Redox Biol
September 2024
The University of Iowa, Iowa City, IA, USA. Electronic address:
In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis.
View Article and Find Full Text PDFTemporary alteration of neuronal network communication is a protective response to redox imbalance that requires GPI-anchored prion protein.
Redox Biol
July 2023
Laboratory of Persistent Viral Diseases, Division of Intramural Research, Rocky Mountain Laboratories, National Institutes of Health, Hamilton, MT, 59840, USA. Electronic address:
Cellular prion protein (PrP) protects neurons against oxidative stress damage. This role is lost upon its misfolding into insoluble prions in prion diseases, and correlated with cytoskeletal breakdown and neurophysiological deficits. Here we used mouse neuronal models to assess how PrP protects the neuronal cytoskeleton, and its role in network communication, from oxidative stress damage.
View Article and Find Full Text PDFDetection and characterization of free oxygen radicals induced protein adduct formation in differentiating macrophages.
Biochim Biophys Acta Gen Subj
May 2023
Department of Biophysics, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic. Electronic address:
Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages.
View Article and Find Full Text PDFExtracellular biomolecular free radical formation during injury.
Free Radic Biol Med
August 2022
The University of Iowa, Iowa City, IA, USA. Electronic address:
Determine if oxidative damage increases in articular cartilage as a result of injury and matrix failure and whether modulation of the local redox environment influences this damage. Osteoarthritis is an age associated disease with no current disease modifying approaches available. Mechanisms of cartilage damage in vitro suggest tissue free radical production could be critical to early degeneration, but these mechanisms have not been described in intact tissue.
View Article and Find Full Text PDFAnalysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates.
Nitric Oxide
January 2022
Amity Institute of Biotechnology, Amity University, Kolkata, 700135, West Bengal, India; Department of Surgery, University of Pittsburgh, Pittsburgh, PA, 15213, USA; Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden. Electronic address:
The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!