Two Dimentional(2D)-Western Blotting Analysis of Clinical Samples Using Fully Automated 2D-Gel Electrophoresis System

We have been studying specific proteins related to the tumorigenesis, malignancy, and chemosensitivity of gliomas using 2D-DIGE and iTRAQ, and so far have identified 69 up-regulated proteins in chemoresistant gliomas. Among them, we focused on vimentin, which shows significant modifications such as phosphorylation and fragmentation in chemoresistant gliomas. To analyze the expression pattern of vimentin in glioma, we established a rapid and sensitive 2-dimentional western blotting method (2D-western) using an auto-2D machine which was recently developed by our group. In 2D-DIGE, 16 vimentin spots with several modifications were identified, and these spots were classified into 4 groups: 53kDa (group 1), 48kDa (group 2), 45kDa (group 3) and 42kDa (group 4). Using auto-2D-western, we confirmed that each group has 3-5 spots with different PIs, and, in total, more than 14 spots were significantly detectable by anti-C-terminal vimentin antibody. Group 4 spots and acidic spots of group 1 could not be detected by anti-N-terminal vimentin antibody, suggesting that vimentin may be cleaved and/or highly phosphorylated at the N-terminal region, respectively. Also, vimentin spots formed after fragmentation induced by caspase, or calpain activation, were detected reproducibly in U373 glioma cells, and these fragmentation patterns were similar to those of chemotherapy-resistant gliomas. Using auto-2D western with antibody cocktails (mixed antibodies against an internal standard and several target proteins), the quantitative studies were quickly performed in a reproducible manner. These results suggest that this auto-2D western system will be useful for the quantitative analysis of not only brain tumors, but also a variety of clinical and biological samples.