Analysis of protein-protein interactions with cTHY28
Stephens, Christopher Brad
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cTHY28 is a nuclear localized phosphoprotein that was previously isloated from chicken lymphocytes during a screening procedure for apoptotic genes; however, the function of this gene product is not well understood. In order to elucidate the cellular function of cTHY28, an immunoprecipitation assay was developed. Using this assay, preliminary data indicated that there was a protein-protein interaction between cTHY28, nucleolin and DNA topoisomerase I (topoI). To further analyze this protein-protein interaction in order to futher characterize cTHY28’s function, a reciprocal co-immunoprecipitation assay was developed. In order to accomplish this goal, the chicken nucleolin and topoI genes were isolated and ligated into a pET28b bacterial overexpression vector. Using this vector system, sufficent quantites of nucleolin and topoI, as well as cTHY28, were generated and served as an antigen source to immunize rabbits for production of anti-cTHY28, anti-nucleolin and anti-topoI antisera. These antisera were characterized using an ELISA (Enzyme Linked Immunosorbent Assay) and western immunoblot analysis. Antibodies isolated from these antisera were used to develop a reciprocal co-immunoprecipitation assay. However, experiments using this assay procedure failed to confirm a cTHY28/nucleolin/topoI protein interaction, although it did demonstrate a protein-protein interaction between nucleolin and topoI.