Naslov
Oxime blot: A novel method for reliable and sensitive detection of carbonylated proteins in diverse biological systems

Autori
Ladouce, Romain ; Combes, Guillaume Fabien ; Trajković, Katarina ; Drmić Hofman, Irena ; Merćep, Mladen

Izvornik
Redox Biology (2213-2317) 63 (2023); 102743, 10

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Amyotrophic lateral sclerosis ; oxidative stress ; alpha synuclein ; superoxide dismutase ; plasma proteins ; quality control ; breast cancer ; disease ; Parkinsons ; damage

Sažetak
Oxidative stress and oxidative protein damage occur in various biological processes and diseases. The carbonyl group on amino acid side chains is the most widely used protein oxidation biomarker. Carbonyl groups are commonly detected indirectly through their reaction with 2, 4-dinitrophenylhydrazine (DNPH) and subsequent labeling with an anti-DNP antibody. However, the DNPH immunoblotting method lacks protocol standardiza-tion, exhibits technical bias, and has low reliability. To overcome these shortcomings, we have developed a new blotting method in which the carbonyl group reacts with the biotin-aminooxy probe to form a chemically stable oxime bond. The reaction speed and the extent of the carbonyl group derivatization are increased by adding a p-phenylenediamine (pPDA) catalyst under neutral pH conditions. These improvements are crucial since they ensure that the carbonyl derivatization reaction reaches a plateau within hours and increases the sensitivity and robustness of protein carbonyl detection. Furthermore, derivatization under pH-neutral conditions facilitates a good SDS-PAGE protein migration pattern, avoids protein loss by acidic precipitation, and is directly compatible with protein immunoprecipitation. This work describes the new Oxime blot method and demonstrates its use in detecting protein carbonylation in complex matrices from diverse biological samples.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti, Kliničke medicinske znanosti

POVEZANOST RADA