Bimolecular Fluorescence Complementation is a modern method to monitor protein-protein interactions in live cells. The objective of this... Show moreBimolecular Fluorescence Complementation is a modern method to monitor protein-protein interactions in live cells. The objective of this project is to employ this method for deciphering signaling complexes involved in androgen receptor mediated cell death. Bcl-2 and Bax were selected to serve as initial experimental targets since these proteins were known to interact with each other. In this method, split EGFP consisting of 1-157 amino acid N-terminal fragment was fused to Bcl-2 and 158-238 amino acid Cterminal fragment to that of Bax with a six amino acid linker (SGSGVD) to generate fusion expression cassettes. We tested two combinations of fusion expression cassette pairs NGFP/Bcl-2, Bax/CGFP and NGFP/Bcl-2, CGFP/Bax for a BiFC signal by cotransfecting them in Human Embryonic Kidney 293T cells. Our results show that fusion proteins NGFP/Bcl-2, Bax/CGFP and CGFP/Bax were well expressed as evident by the Western blots. But the efficiency of BiFC formation was better for NGFP/Bcl-2 and CGFP/Bax fusion proteins combination than NGFP/Bcl-2 and Bax/CGFP fusion proteins. Therefore indicate that the fluorescence complex formation imparting the BiFC signal is specific for Bcl-2 and Bax interaction. Though the BiFC signal obtained was brighter for image capturing but the efficiency was very low, only few fluorescing cells were detected under the fluorescence microscope. Despite this weakness, BiFC is still a promising technique for studying protein-protein interactions in living cells and at single cell level. M.S. in Biology, July 2011 Show less
