Standardization of protocol for Immobiline 2-D PAGE and construction of 2-D PAGE protein database on World Wide Web home page

Organismic aging appears in the results of functional alteration of tissue-constituent cells. The replicative life span of normal human diploid fibroblasts in cell culture has been realized as a typical model of the research on mitotic cell aging. From the above premises, a new project has started in our institute for establishing my own age-related protein database of human diploid fibroblasts upon a 2-D PAGE technique. The protocol of 2-D PAGE with horizontal IPG-IEF and vertical SDS-PAGE was standardized including whole procedures from cell harvest through spot quantification, since the reproducibility of 2-D gel pattern depended on the procedure. The master 2-D gel pattern of human diploid fibroblasts was generated by merging skin fibroblast-specific spots into the standard 2-D gel pattern of lung fibroblasts in an image analyzing software, because fibroblasts showed tissue specificity. The master 2-D gel pattern with spot identification numbers and the database of age-dependent protein alteration were presented on my home page for free access through the Internet computer network.