Identification of oxidized proteins based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunochemical detection, isoelectric focusing, and microsequencing.
It has been demonstrated that an age-associated increase in protein oxidation is a highly selective rather than random phenomenon. Addition of carbonyl groups is a widely used marker for the detection of oxidative damage to the proteins. Carbonyls can be detected immunochemically using antibodies against dinitrophenylhydrazine (DNPH), a reagent that specifically reacts with protein carbonyl groups. In this paper, a procedure for the identification of oxidized proteins during aging or under pathological conditions is described. Protein samples were treated with DNPH and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunochemical detection. Proteins exhibiting positive immunostain were then purified by SDS-PAGE followed by isoelectric focusing. The focused protein band was further concentrated by another run of SDS-PAGE and transferred onto Immobilon-P membrane. The identity of the protein was revealed by automated Edman microsequencing and a computer database search. This method is successfully demonstrated for the identification of housefly arginine kinase, a cytosolic protein that is involved in the energy metabolism of insect muscle cells.
Yan
L J
LJ
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA.
Orr
W C
WC
Sohal
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007120:Immunochemistry / Q000379:methods*; D007525:Isoelectric Focusing; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D017421:Sequence Analysis / Q000379:methods*; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling / Q000379:methods
D008567:Membranes, Artificial; D009124:Muscle Proteins; D010659:Phenylhydrazines; D011506:Proteins; C004787:2,4-dinitrophenylhydrazine; D012967:Sodium Dodecyl Sulfate