title: An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.
abstract: Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73Eprotein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73Eproteins) as a biological example.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago.
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Hsieh
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago.
authors.forename: Amel
authors.initials: A
authors.lastname: Alqadah
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago; chioufen.chuang@gmail.com.
authors.forename: Chiou-Fen
authors.initials: CF
authors.lastname: Chuang
mesh_terms: D001483:Base Sequence; D004247:DNA; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D005456:Fluorescent Dyes; D009841:Oligonucleotides
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA