Oxidative stress is commonly defined as a condition under which the generation of reactive oxygen species (ROS) within a cellular system exceeds the buffering capacity of endogenous antioxidant defenses. This leads to oxidative damage to lipids, DNA and proteins, and has been linked to aging, cancer and other diseases. One of the most commonly used markers of oxidative stress is carbonylation of proteins.

The RayBio® Oxidation (Carbonyl) Detection Kit utilizes the ‘biotin probes’ method to biotin label carbonyl groups of oxidized proteins. The biotin labelled proteins can then be detected by western blotting. In this method, the biotin probes first react with carbonyl groups of oxidized proteins to form the unstable Schiff bases, which are then further reduced to more stable amines. The biotinylated proteins can then be detected by western blotting. Furthermore, the oxidation sites can be mapped using mass spectrometry after selectively isolating the biotinylated proteins/peptides through avidin resin.

1. 20% (v/v) trichloroacetic acid (TCA) solution, ice-cold
2. 1:1 (v/v) ethanol:ethyl acetate
3. Desalting column or dialysis membrane
4. 1.5 mL microcentrifuge tubes
5. 15 ml tubes (polypropylene)
6. 10 ml graduated cylinders (X2)
7. Benchtop centrifuge and microcentrifuge (4°C)
8. Distilled or deionized water
9. Precision pipettes to deliver 2 µl to 1 ml volumes
10. Adjustable 1-25 ml pipettes for reagent preparation

1. Prepare all reagents and samples as instructed.
2. Label samples with Oxidation Labeling Reagent.
3. Add Oxidation Stabilizing Reagent to samples.
4. Precipitate proteins with ice-cold 20% TCA.
5. Wash protein pellet twice with 1:1 (v/v) ethanol:ethyl acetate.
6. Resuspend protein pellets in Oxidation Buffer B.