全自動二次元電気泳動装置を用いたタンパク質の分離とリン酸化パターンの解析

抄録

For the personal diagnosis, we developed an easy, highly reproducible and fully automated two-dimensional (2D) gel electrophoresis system. Prior to using the clinical samples, the performance of this system was evaluated by using the samples prepared from mouse tissues. Also, a cell-based tumorigenesis model was constructed by treating the normal phenotype mouse fibroblast, NIH3T3, with a tumor promoter, TPA, which induces the tumor-like signals. The transition of cellular protein phosphorylation status by treatment with TPA was further investigated by phosphoprotein staining and western blotting using antibodies against some major proteins in cell growth signaling pathway. Analyses of positions and shapes of some selected protein spots revealed that our 2D system had resolution comparable to commercial minigel systems. We also investigated the transition of the protein phosphorylation status by 2D-western blotting in NIH3T3 tumorigenesis model. Because p44 and p42 MAPK has two phosphorylation sites each, their spots are shifted to acidic side according to the number of phosphorylation. After treatment with TPA, the antibody against p44 and p42 MAPK detected 6 spots (3 spots each), while no treated samples showed only 2 spots (1 spot each). Furthermore, when the two images of western blots were overlapped, the spots located in alkaline side was shown to be also overlapped. Taken together, these data indicated that our 2D system had high reproducibility and resolution enough to separate one amino acid phosphorylation. We further constructed a cell-based inflammatory response model. Differentiated human monocytic leukemia cell line, THP-1, was treated with LPS. We will present the investigation resluts concerning the transition of cellular protein status by western blotting using antibodies against proteins related to immune response.