caa a22 4500 397557388 CHVBK 20180308164806.0 cr unu---uuuuu 161202e199604 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021306 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021306 Analysis of Protein Carbonyls with 2,4-Dinitrophenyl Hydrazine and Its Antibodies by Immunoblot in Two-Dimensional Gel Electrophoresis [Elektronische Daten] [Akihiro Nakamura, Sataro Goto] Protein carbonyls are reported to increase in aging and in pathologies such as Alzheimer's disease and ischemic injury. Detailed study of this important issue has, however, been hampered by lack of an appropriate method to identify individual carbonylated proteins. We describe here an immunoblot method to investigate protein carbonyls reactive to 2,4-dinitrophenyl hydrazine. Rabbit polyclonal antibodies against 2,4-dinitrophenyl hydrazine were used to study the proteins derivatized by the reagent in one- or two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting. More than 25 proteins with high carbonyl contents were clearly demonstrated in two-dimensional immunoblot of rat tissue soluble proteins. The method could detect concentrations as low as 1 pmol of carbonyls. The signals were mostly abolished by prior treatment of tissue proteins with sodium borohydride to reduce carbonyls. Fragments generated by V8 protease digestion of a single protein exhibited signal intensities of varying degrees, indicating that carbonyla-tion is not uniform in different amino acid sequences. Proteins treated with glucose or aldehydes gave rise to positive signals, suggesting that the finding of carbonyls in tissue proteins is not necessarily an indication of direct oxidation of side chains of amino acid residues. © by the Journal of Biochemistry Regular Papers nationallicence aging nationallicence immunoblot nationallicence protein oxidation nationallicence protein carbonyl nationallicence rat nationallicence Nakamura Akihiro Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Miyama, Fiwiabashi, Chiba 274 aut Goto Sataro Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Miyama, Fiwiabashi, Chiba 274 aut The Journal of Biochemistry Oxford University Press 119/4(1996-04), 768-774 0021-924X 119:4<768 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021306 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021306 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nakamura Akihiro Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Miyama, Fiwiabashi, Chiba 274 aut NATIONALLICENCE 700 1- Goto Sataro Department of Biochemistry, School of Pharmaceutical Sciences, Toho University, Miyama, Fiwiabashi, Chiba 274 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/4(1996-04), 768-774 0021-924X 119:4<768 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford