Mikulić Filip Luka, Merćep Viktor, Finek Marcela, Merćep Mladen
Department of Emergency Medicine of the Krapina-Zagorje County, Krapina, Croatia.
School of Medicine, University of Zagreb, Zagreb, Croatia.
Bio Protoc. 2025 Aug 5;15(15):e5401. doi: 10.21769/BioProtoc.5401.
Oxidative protein damage is important in various biological processes and age-related diseases. Protein carbonylation is the predominant and most frequently studied form of protein oxidation. It is most frequently detected following its derivatization with 2,4-dinitrophenylhydrazine (DNPH) hapten, followed by its detection with an anti-DNP antibody. However, when used to detect protein carbonylation by western blotting, this method suffers from diminished sensitivity, distortion of protein migration patterns, and unsatisfactory representation of low-abundance proteins. This is due to the poor solubility of DNPH in typical buffer solutions, the acidic protein precipitation due to the use of strong acid for its dissolution, the instability in solution, and the distorted protein migration patterns introduced by an additional salt content generated by the required pH adjustment prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To address the DNPH method limitations, a new Oxime blot technique was developed. This method is based on forming the stable oxime bonds between the protein carbonyl groups and biotin-aminooxy probe in the presence of a p-phenylenediamine (pPDA) catalyst at neutral pH conditions. The derivatization reaction reaches a plateau within 3 h. It ensures efficient and complete derivatization of carbonylated proteins, which are separated by SDS-PAGE without additional manipulation and detected with avidin-HRP and enhanced chemiluminescence (ECL) in western blotting. The Oxime blot protocol allows researchers to reliably and sensitively detect carbonylated proteins and provides a valuable tool for studying oxidative stress in diverse biological settings. Key features • This method enables the sensitive and reliable detection of protein carbonylation in various biological samples. • The chemically stable oxime bond forms quickly and efficiently, reaching its plateau level after 3 h, enabling relative carbonylation quantification. • Carbonylation derivatization at low salt content and neutral pH ensures good SDS-PAGE protein migration without any protein loss. • This method integrates well with detecting specific protein carbonylation following its immunoprecipitation.
氧化蛋白质损伤在各种生物过程和与年龄相关的疾病中都很重要。蛋白质羰基化是蛋白质氧化的主要且研究最频繁的形式。它最常通过与2,4-二硝基苯肼（DNPH）半抗原衍生化后，再用抗DNP抗体进行检测。然而，当用于通过蛋白质印迹法检测蛋白质羰基化时，该方法存在灵敏度降低、蛋白质迁移模式失真以及低丰度蛋白质呈现不令人满意的问题。这是由于DNPH在典型缓冲溶液中的溶解度差、使用强酸溶解导致酸性蛋白质沉淀、溶液中的不稳定性以及在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）之前进行所需的pH调节所产生的额外盐分导致蛋白质迁移模式失真。为了解决DNPH方法的局限性，开发了一种新的肟印迹技术。该方法基于在对苯二胺（pPDA）催化剂存在下，在中性pH条件下蛋白质羰基与生物素-氨氧基探针之间形成稳定的肟键。衍生化反应在3小时内达到平稳状态。它确保了羰基化蛋白质的高效、完全衍生化，这些蛋白质无需额外操作即可通过SDS-PAGE分离，并在蛋白质印迹法中用抗生物素蛋白-辣根过氧化物酶（avidin-HRP）和增强化学发光（ECL）进行检测。肟印迹方案使研究人员能够可靠且灵敏地检测羰基化蛋白质，并为研究不同生物环境中的氧化应激提供了一个有价值的工具。关键特性 • 该方法能够灵敏可靠地检测各种生物样品中的蛋白质羰基化。 • 化学稳定的肟键快速有效形成，3小时后达到平稳水平，能够进行相对羰基化定量。 • 在低盐含量和中性pH下进行羰基化衍生化可确保良好的SDS-PAGE蛋白质迁移，且无任何蛋白质损失。 • 该方法与免疫沉淀后检测特定蛋白质羰基化很好地整合。
