Functional Analysis of Proteins regulated by Ubiquitin Ligase

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We have tried to identify physiological substrates of ubiquitin ligase by establishment of comprehensive-analytical method for detection of difference between before and after genetic engineering in knock-out mice.First, we purified ubiquitinated protein specifically by anti-ubiquitin antibody. Ubiquitin is an abundant protein and exist as free form (not conjugated or binding form in mammalian cells. By affinity-chromatography using a conventional antibody, we recovered a free-ubiqitin mainly, and could not collect ubiquitin conjugated proteins. For improvement of the efficiency of collection, we assesed many kinds of antibodies and finally decided to use the FK2 monoclonal antibody, which binds only substrate-conjugated ubiqitin specifically. We succeeded in concentrating ubiquitinated proteins by affinity-chromatography using FK2 binding column.For comparison of proteins between knock-out mice and wild type mice, we have to choose the most adequate tissue and the most effective method for cell-extraction. We identified the tissue which expressed F-box protein we were interested in by in-situ hybridization.Based on these basic informations, we prepared Fbw7-deficient-embryonic fibroblasts and also wild-type fibroblast, and extracted whole cell lysate from each samples. Cell lysate loaded on column packed FK2-antibody conjugated beads for concentration of ubiquitinated proteins. We plan to compare the difference of the ubiquitinated proteins between Fbw7-deficient and wild-type embryonic fibroblasts.