Reliable and Sensitive Detection of Carbonylated Proteins by Oxime Blot
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, f...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Bio-protocol LLC
2025-08-01
|
| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5401&type=0 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | 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. |
|---|---|
| ISSN: | 2331-8325 |