Determining transcription factor-promoter interaction by electrophoretic mobility shift assay

The organization and use of information on the DNA molecules is closely related with protein binding. DNA binding proteins have important roles in all steps of DNA packaging, transcription, replication and repair. Understanding the nature of DNA-protein interactions are important for analyzing genetic processes.DNaseI foot printing, nitrocellulose filter binding and ChIP methods are used for direct detection of DNA-protein interactions. Electrophoretic mobility shift assay (EMSA) is a fast and sensitive method for determination of nucleic acids-DNA interactions. Although EMSA is a radioactive method, it can also be used with non-radioactive labeling.In our work, we tried to optimize a non-radioactive based EMSA method in our laboratory. Wnt5b, Fzd4, Fzd5 and Fzd7 gene promoters were used in our work. The transcription factors which bind to promoter regions of these genes were determined with bioinformatics methods. We worked with three transcription factors which bind to all of the four gene promoters. These are the Sp1, Egr-1 and KLF transcription factors which were determined by Genomatics genome analyzing software.We optimized the non-radioactive EMSA method for our laboratory and we plan to use this method for our future projects to explore DNA-Protein interactions.Key Words: EMSA, protein-DNA interaction, Sp1 transcription factor