Proceeding of Veterinary and Animal Science Days 2016, 8th- 10th June, Milan, Italy 

HAF © 2013 

Vol. III, No. 1s  ISSN: 2283-3927 

   

 

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Keywords 

Dog, western blotting, carbonyl 

group proteins, oxidative stress, 

oxidants. 

 

CORRESPONDING AUTHOR 

Beatrice Ruggerone 

beatrice.ruggerone@unimi.it 

 

JOURNAL HOME PAGE 

riviste.unimi.it/index.php/haf 

Analytical aspects of protein carbonyl group 

in oxidized canine serum. 

 

B. Ruggeronea*, G. Colombob, S. Paltrinieria 

 

 

a Department of Veterinary Medicine, University of Milan, Via Celoria, 10 -20133 
Milan, Italy 

b Department of Biosciences, University of Milan, via Celoria 26, 20133 Milan, Italy 

 

Abstract 

Oxidative stress (OS) is an imbalance between oxidants and anti-oxidants and plays an important role 

in the aetiology and/or the progression of several diseases. Protein carbonyl (PCO) groups are so far 

used as biomarkers of OS in humans (Colombo et al, 2015). 

The aim of our study was to investigate whether PCOs are present in canine serum and if they can be 

measured spectrophotometrically using a method not yet validated in dogs. 

The presence of PCO was investigated by Western blotting after separation by SDS-PAGE of serum at 

different dilutions (either before or after oxidation with 10% cigarette smoke extract). Protein labelling 

with 2,4-Dinitrophenylhydrazine (DNPH, Brady's reagent) was followed by a two-steps incubation with 

primary anti-dinitrophenyl-KLH antibodies (rabbit IgG fraction) and secondary goat anti-rabbit IgG, HRP 

conjugate. Signal was developed with Enhanced Chemiolumionescence (ECL).  

Serum PCO were quantified using a commercially available assay (Protein Carbonyl Content Assay Kit - 

Abcam, UK), based on derivatization of proteins with 1,4-dinitrophenylhydrazine (DNPH) and 

subsequent formation of protein-conjugated dinitrophenylhydrazones (DNPs) with a peak absorbance 

at 366 nm. 

Western blot showed an evident band of apparent MW of 69 kDa, consistent with carbonylated dog 

serum albumin. The spectrophotometric assay failed to demonstrate any signal at 366 nm using the 

manufacturer’s instructions or modified protocols.   

This study demonstrated that PCO are present in oxidized canine serum. However, the 

spectrophotometric assay employed in this study is not enough sensitive to detect PCOs.  Further 

studies are needed to assess whether this depends on the poor re-solubilisation of DNPs, or on the low 

concentration of PCOs in dogs. 
 

References 

Colombo, G., Clerici, M., Garavaglia, M.E., Giustarini, D., Rossi, R., Milzani, A., Dalle-Donne, I., 2015.  A step-by-step 

protocol for assaying protein carbonylation in biological samples. J Chromatogr B.  

 

103, 325-330.  

http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en