PMID- 10729190
OWN - NLM
STAT- MEDLINE
DCOM- 20000426
LR  - 20161124
IS  - 0003-9861 (Print)
IS  - 0003-9861 (Linking)
VI  - 376
IP  - 1
DP  - 2000 Apr 1
TI  - Adaptation of protein carbonyl detection to the requirements of proteome analysis 
      demonstrated for hypoxia/reoxygenation in isolated rat liver mitochondria.
PG  - 59-65
AB  - The key technique in proteome analysis is high-resolution two-dimensional (2D) 
      electrophoretic separation of proteins from biological samples. This method 
      combines isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel 
      electrophoresis (SDS-PAGE). Derivatization of protein carbonyls with 2, 
      4-dinitrophenylhydrazine (DNPH) and subsequent anti-dinitrophenyl (DNP) 
      immunoblotting is widely used for the detection of oxidatively modified proteins. 
      In previous studies on adapting this method to 2D electrophoresis the 
      derivatization step was carried out before and after the 2D procedure, resulting 
      in an altered spot pattern and high background staining, respectively. The aim of 
      the present experiments was to develop a method for protein derivatization with 
      DNPH between the IEF and the SDS-PAGE steps. Mitochondria were exposed to 10 min 
      hypoxia and 5 min reoxygenation. After IEF using immobilized pH gradients the gel 
      strips were incubated in DNPH/trifluoroacetic acid/SDS for 20 min and 
      neutralized, and SDS-PAGE was performed. Proteins were either stained with 
      Coomassie dye or subjected to Western blotting using anti-DNP IgG. Gels and blots 
      were scanned and matched to a master gel, and the relative carbonyl content of 
      each spot was calculated and compared for five experiments. Importantly, the spot 
      patterns in DNPH-treated and untreated gels were not different. Protein carbonyls 
      could be detected in 59 of the 125 matched spots. Although there was no 
      significant increase in the total protein carbonyl content after 
      hypoxia/reoxygenation, eighteen 2D spots exhibited an increase in carbonyl 
      content. However, most protein spots did not show a change or even a decline (4 
      spots) in protein carbonyls.
CI  - Copyright 2000 Academic Press.
FAU - Reinheckel, T
AU  - Reinheckel T
AD  - Department of Experimental Surgery, University of Magdeburg, Magdeburg, D-39120, 
      Germany. reinheckel@mm11.ukl.unifreiburg.de
FAU - Korn, S
AU  - Korn S
FAU - Mohring, S
AU  - Mohring S
FAU - Augustin, W
AU  - Augustin W
FAU - Halangk, W
AU  - Halangk W
FAU - Schild, L
AU  - Schild L
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Arch Biochem Biophys
JT  - Archives of biochemistry and biophysics
JID - 0372430
RN  - 0 (Indicators and Reagents)
RN  - 0 (Phenylhydrazines)
RN  - 0 (Proteins)
RN  - 0 (Proteome)
RN  - 1N39KD7QPJ (2,4-dinitrophenylhydrazine)
RN  - S88TT14065 (Oxygen)
SB  - IM
MH  - Animals
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Evaluation Studies as Topic
MH  - Hydrogen-Ion Concentration
MH  - Hypoxia/*metabolism
MH  - In Vitro Techniques
MH  - Indicators and Reagents
MH  - Isoelectric Focusing
MH  - Male
MH  - Mitochondria, Liver/*metabolism
MH  - Oxygen/metabolism
MH  - Phenylhydrazines
MH  - Proteins/*chemistry/*metabolism
MH  - Proteome/*analysis/*metabolism
MH  - Rats
MH  - Rats, Wistar
EDAT- 2000/03/24 09:00
MHDA- 2000/04/29 09:00
CRDT- 2000/03/24 09:00
PHST- 2000/03/24 09:00 [pubmed]
PHST- 2000/04/29 09:00 [medline]
PHST- 2000/03/24 09:00 [entrez]
AID - S0003-9861(99)91680-6 [pii]
AID - 10.1006/abbi.1999.1680 [doi]
PST - ppublish
SO  - Arch Biochem Biophys. 2000 Apr 1;376(1):59-65. doi: 10.1006/abbi.1999.1680.