Electrophoretic Mobility-Shift and Super-Shift Assays for Studies and Characterization of Protein–DNA Complexes

Abstract

Gene expression is in part regulated by transcription factors that bind specific sequence motifs in genomic DNA. Transcription factors cooperate with the basal machinery to upregulate or downregulate transcription. Experimental data have revealed the importance of interactions among members of distinct families of transcription factors to form complexes that regulate gene expression. Thus, a full characterization of protein–DNA complexes is essential to understanding of gene regulation in a more complex cellular environment. Electrophoretic mobility shift assay (EMSA) is a powerful technique to resolve nucleic acid–protein complexes formed with transcription factors in nuclear extracts. Herein is described how EMSA and super-shift assays were used to characterize several complexes produced from binding of transcription factors to a regulatory DNA sequence upstream from the promoter region of the human NF-IL6 gene.