Technology of the month
Category
Protocols / Protein biochemistry
Main applications
Carbonylation is an irreversible oxidative modification that can alter the physicochemical properties and function of proteins. Protein carbonylation occurs during oxidative stress and increases with aging. It is widely accepted that the accumulation of oxidatively modified macromolecules contributes to cellular damage, aging, and the initiation and progression of age-related diseases. The measurement of protein carbonylation in biological samples can contribute to our understanding of protein biochemistry, shed light on oxidative stress-related mechanisms in various diseases, and provide diagnostic and prognostic markers. The presented oxime blot approach represents a novel method for the precise detection of protein carbonylation in recombinant or immunoprecipitated proteins and in cell lysates.
The technology
In this new blotting method for the detection of protein carbonyls, the carbonyl group of an oxidized protein reacts with a biotin-aminooxy probe to form a chemically stable oxime bond. The reaction is catalyzed by p-phenylenediamine (pPDA). The proteins are then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a membrane, followed by carbonyl detection using a fluorescent dye-conjugated streptavidin probe or horseradish peroxidase. The method is more accurate and user-friendly than the existing 2,4-dinitrophenylhydrazine-based method.
Requirements
- Standard Equipment for SDS-PAGE and Western Blotting
- Derivatizing Agent (Biotin-Aminooxy)
- Catalyst (p-phenylenediamine)
- Streptavidin conjugates (either fluorescent or luminescent probe)
Future perspectives
Oxidation of adhesion G protein-coupled receptors (aGPCRs) is understudied, although aGPCR proteins can be regulated by their redox state. Therefore, the aGPCR field could benefit from applying this method to assess the carbonylation status of aGPCR proteins and proteins involved in related pathways. Furthermore, understanding the impact of oxidative modifications on aGPCRs proteins has potential therapeutic implications.
Contact person
Combes Guillaume (Postdoctoral researcher)
Mediterranean Institute for Life Sciences (MedILS)
Split, Croatia
Links to webpages
https://www.medils.org/
https://www.medils.org/research/scientists?cid=1709
Ladouce R, Combes GF, Trajković K, Drmić Hofman I, Merćep M. Oxime blot: A novel method for reliable and sensitive detection of carbonylated proteins in diverse biological systems. Redox Biol. 2023 Jul;63:102743. doi: 10.1016/j.redox.2023.102743. Epub 2023 May 12. PMID: 37207613; PMCID: PMC10206197.
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