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, J; Gao, Y; Ju, Y; Yang, J; Wu, Q; Zhang, J; Du, X; Wang, Z; Song, Y; Li, H; Luo, X; Ren, F; Li, J; Chen, Y; Wang, L; Xu, H; Liu, X; Wang, J; Zhang, Y; Cai, Y; Cui, Y; Qian, X; He, F; Li, M; Sun, Q-H
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