title: Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins.
abstract: 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.
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, PR China.
authors.forename: Jianen
authors.initials: J
authors.lastname: Gao
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Ju
authors.forename: Jinju
authors.initials: J
authors.lastname: Yang
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.forename: Jinchao
authors.initials: J
authors.lastname: Zhang
authors.forename: Xuemei
authors.initials: X
authors.lastname: Du
authors.forename: Zhaoqing
authors.initials: Z
authors.lastname: Wang
authors.forename: Yingbo
authors.initials: Y
authors.lastname: Song
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Xiaotong
authors.initials: X
authors.lastname: Luo
authors.forename: Feiyao
authors.initials: F
authors.lastname: Ren
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.forename: Lihong
authors.initials: L
authors.lastname: Wang
authors.forename: Han
authors.initials: H
authors.lastname: Xu
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Liu
authors.forename: Jinlan
authors.initials: J
authors.lastname: Wang
authors.forename: Yangjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yun
authors.initials: Y
authors.lastname: Cai
authors.forename: Yufang
authors.initials: Y
authors.lastname: Cui
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Qi-Hong
authors.initials: QH
authors.lastname: Sun
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D024101:Mitochondrial Proteins; D002222:Carbamoyl-Phosphate Synthase (Ammonia)