creator Chayanam, Sudharani advisor Xiang, Jialing Bimolecular 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. Submitted by Lance Garrison (garrlanc@my.dom.edu) on 2012-03-07T03:05:57Z No. of bitstreams: 2 Title Page.pdf: 124729 bytes, checksum: c2034ce124b0c967c95347e74464bb31 (MD5) MS thesis IIT July 2011_Chayanam.pdf: 1433343 bytes, checksum: 800f24dc8d7bf49264893bafca0ff2f7 (MD5) Made available in DSpace on 2012-03-07T03:05:57Z (GMT). No. of bitstreams: 2 Title Page.pdf: 124729 bytes, checksum: c2034ce124b0c967c95347e74464bb31 (MD5) MS thesis IIT July 2011_Chayanam.pdf: 1433343 bytes, checksum: 800f24dc8d7bf49264893bafca0ff2f7 (MD5) Previous issue date: 2011-07 M.S. in Biology, July 2011 2011-07 2011-07 http://hdl.handle.net/10560/2549 en Thesis born digital application/pdf In Copyright http://rightsstatements.org/page/InC/1.0/ Restricted Access BIOL / Biology Illinois Institute of Technology Affiliated department