Protein-Deoxyribonucleic Acid Interactions Linked to Gene Expression: Electrophoretic Mobility Shift Assay
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The electrophoretic mobility shift assay (EMSA) determines sequence-specific protein-deoxyribonucleic acid (DNA) interactions (e.g., transcription factors with cognate regulatory elements). This assay is based on reduced electrophoretic mobilities of protein/DNA complexes through a nondenaturing polyacrylamide gel compared with unbound DNA fragments or double-stranded oligonucleotides. If multiprotein complexes bind, the migration rate slows further with each additional protein. Oligonucleotide competition and antibody binding controls provide further evidence of binding specificity and protein identity. In competition experiments, unlabeled (cold) DNA or the same oligonucleotide (being used as a probe) with wild-type or mutated sequences of binding site or other unrelated binding sequences are added to the reaction. Interactions between the binding protein and the unlabeled related sequences would decrease the band intensity of the previously shifted complexes, whereas mutated or unrelated binding sequences will not change the intensity of the previously shifted complexes (an example is shown in Fig. 1 ).
Fig. 1. Identification of regulatory protein interactions with promoter elements. Sequence-specific protein-DNA interactions were evaluated at the C/EBP site in the rat Osteocalcin promoter (A) . Oligonucleotides (30 bp) representing wild-type (WT) and mutant (MT) sequences for C/EBP site were gel purified. WT probe was incubated with 6 μg of HeLa nuclear extracts and increasing concentrations (0, 12.5, 25, 50, and 100�) of either unlabeled WT or MT oligonucleotides. Formation of two closely migrating C/EBP specific complexes is indicated. Competition studies demonstrate sequence-specific protein-DNA binding, as the disappearance of complexes is observed only with the addition of WT but not MT competitors. RUNX proteins are the major component of the transcription factor complex binding to Gallus BSP promoter (B) . Electrophoretic mobility antibody supershift analyses are shown for Gallus BSP promoter fragment containing recognition sequences for Runx factors. Nuclear extracts were pre-incubated with 1 μL of Runx antibodies for 20 min at 37�C followed by protein-DNA binding reaction (20 min at room temperature) with the addition of probe. Lanes 1 to 5 represent antibody controls for each site without nuclear extract indicating that antiserum alone does not react with the probe. Lane 6, the control, has 6 μg of nuclear extract without antibodies. Lanes 7–10 show the supershifts of the Runx complex with the Cbfβ, RUNX1, RUNX3, and RUNX2 antibody. The complex formed with ROS17/2.8 nuclear extract is completely supershifted in the presence of antibody specific to RUNX2/Cbfa1 (lane 10). Weak supershifts are also observed using antibodies specific to Cbfβ (a transactivator partner protein for Runx factors) and RUNX1 (lanes 7 and 8, respectively).
