Kondo Tadashi, Hirohashi Setsuo
Proteome Bioinformatics Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Nat Protoc. 2006;1(6):2940-56. doi: 10.1038/nprot.2006.421.
Proteome data combined with histopathological information provides important, novel clues for understanding cancer biology and reveals candidates for tumor markers and therapeutic targets. We have established an application of a highly sensitive fluorescent dye (CyDye DIGE Fluor saturation dye), developed for two-dimensional difference gel electrophoresis (2D-DIGE), to the labeling of proteins extracted from laser microdissected tissues. The use of the dye dramatically decreases the protein amount and, in turn, the number of cells required for 2D-DIGE; the cells obtained from a 1 mm2 area of an 8-12 microm thick tissue section generate up to 5,000 protein spots in a large-format 2D gel. This protocol allows the execution of large-scale proteomics in a more efficient, accurate and reproducible way. The protocol can be used to examine a single sample in 5 d or to examine hundreds of samples in large-scale proteomics.
蛋白质组数据与组织病理学信息相结合，为理解癌症生物学提供了重要的新线索，并揭示了肿瘤标志物和治疗靶点的候选物。我们已将一种为二维差异凝胶电泳（2D-DIGE）开发的高灵敏度荧光染料（CyDye DIGE Fluor饱和染料）应用于激光显微切割组织提取的蛋白质标记。该染料的使用显著减少了蛋白质用量，进而减少了2D-DIGE所需的细胞数量；从8-12微米厚的组织切片1平方毫米区域获得的细胞，在大型二维凝胶中可产生多达5000个蛋白质斑点。该方案能够以更高效、准确和可重复的方式进行大规模蛋白质组学研究。该方案可用于在5天内检测单个样本，或用于大规模蛋白质组学中检测数百个样本。
