Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins.
Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.
Gao
Jianen
J
Beijing Institute of Radiation Medicine, Beijing, PR China.
Gao
Yuan
Y
Ju
Yanfang
Y
Yang
Jinju
J
Wu
Qiong
Q
Zhang
Jinchao
J
Du
Xuemei
X
Wang
Zhaoqing
Z
Song
Yingbo
Y
Li
Hui
H
Luo
Xiaotong
X
Ren
Feiyao
F
Li
Jian
J
Chen
Yong
Y
Wang
Lihong
L
Xu
Han
H
Liu
Xiaolan
X
Wang
Jinlan
J
Zhang
Yangjun
Y
Cai
Yun
Y
Cui
Yufang
Y
Qian
Xiaohong
X
He
Fuchu
F
Li
Ming
M
Sun
Qi-Hong
QH
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D000936:Antigen-Antibody Complex; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000276:immunology / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D015723:Gene Library; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D047468:Immunoprecipitation; D008930:Mitochondria, Liver / Q000276:immunology*; D024101:Mitochondrial Proteins / Q000276:immunology / Q000378:metabolism*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D024101:Mitochondrial Proteins; D002222:Carbamoyl-Phosphate Synthase (Ammonia)