For COVID-19 vaccine updates, please review our information guide. For patient eligibility and scheduling availability, please visit VaccineTogetherNY.org.

A FRET-based method to study protein thiol oxidation in histological preparations.

TitleA FRET-based method to study protein thiol oxidation in histological preparations.
Publication TypeJournal Article
Year of Publication2008
AuthorsMastroberardino PG, Orr AL, Hu X, Na HMee, J Greenamyre T
JournalFree Radic Biol Med
Volume45
Issue7
Pagination971-81
Date Published2008 Oct 01
ISSN0891-5849
KeywordsAnimals, Blotting, Western, Disulfides, Fluorescence Resonance Energy Transfer, Humans, Immunohistochemistry, Microscopy, Confocal, Oxidation-Reduction, Proteins, Sulfhydryl Compounds
Abstract

Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique-which can be used in association with standard immunological staining procedures-to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool for providing further understanding of oxidative imbalance, a phenomenon common to many diseases.

DOI10.1016/j.freeradbiomed.2008.06.018
Alternate JournalFree Radic. Biol. Med.
PubMed ID18620047
PubMed Central IDPMC2605956
Grant ListK99 ES016352-02 / ES / NIEHS NIH HHS / United States
K99 ES016352 / ES / NIEHS NIH HHS / United States
K99-ES016352 / ES / NIEHS NIH HHS / United States
K99 ES016352-01 / ES / NIEHS NIH HHS / United States
U54 ES012068 / ES / NIEHS NIH HHS / United States
U54-ES012068 / ES / NIEHS NIH HHS / United States