Analysis of protein carbonyls with 2,4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis

Akihiro Nakamura, S. Goto

Research output: Contribution to journalArticle

145 Citations (Scopus)

Abstract

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 carbonylation 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.

Original languageEnglish
Pages (from-to)768-774
Number of pages7
JournalJournal of Biochemistry
Volume119
Issue number4
Publication statusPublished - Apr 1996

Fingerprint

hydrazine
Electrophoresis, Gel, Two-Dimensional
Electrophoresis
Gels
Antibodies
Proteins
Tissue
Carbonylation
Amino Acids

Keywords

  • Aging
  • Immunoblot
  • Protein carbonyl
  • Protein oxidation
  • Rat

ASJC Scopus subject areas

  • Biochemistry

Cite this

Analysis of protein carbonyls with 2,4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis. / Nakamura, Akihiro; Goto, S.

In: Journal of Biochemistry, Vol. 119, No. 4, 04.1996, p. 768-774.

Research output: Contribution to journalArticle

@article{fbd377088fcb4a369ccaa3b2a8654b3f,
title = "Analysis of protein carbonyls with 2,4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis",
abstract = "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 carbonylation 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.",
keywords = "Aging, Immunoblot, Protein carbonyl, Protein oxidation, Rat",
author = "Akihiro Nakamura and S. Goto",
year = "1996",
month = "4",
language = "English",
volume = "119",
pages = "768--774",
journal = "Journal of Biochemistry",
issn = "0021-924X",
publisher = "Oxford University Press",
number = "4",

}

TY - JOUR

T1 - Analysis of protein carbonyls with 2,4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis

AU - Nakamura, Akihiro

AU - Goto, S.

PY - 1996/4

Y1 - 1996/4

N2 - 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 carbonylation 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.

AB - 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 carbonylation 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.

KW - Aging

KW - Immunoblot

KW - Protein carbonyl

KW - Protein oxidation

KW - Rat

UR - http://www.scopus.com/inward/record.url?scp=0029965252&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029965252&partnerID=8YFLogxK

M3 - Article

C2 - 8743580

AN - SCOPUS:0029965252

VL - 119

SP - 768

EP - 774

JO - Journal of Biochemistry

JF - Journal of Biochemistry

SN - 0021-924X

IS - 4

ER -