title: Analysis of In Vitro DNA Interactions of Brassinosteroid-Controlled Transcription Factors Using Electrophoretic Mobility Shift Assay.
abstract: Most signaling cascades ultimately lead to changes in gene expression by modulating the activity of transcription factors (TFs). The electrophoretic mobility shift assay (EMSA) is a simple but powerful in vitro method for investigation of specific protein-DNA interactions. It makes use of the fact that protein-DNA complexes have a lower electrophoretic mobility in gels than free DNA has. The application of labeled probes in combination with unlabeled competitors allows investigation of DNA-binding specificity and identification of binding motifs with single base-pair resolution. Here we describe the application of EMSAs for the study of interactions of the brassinosteroid-regulated TFs, BRASSINAZOLE-RESISTANT1, (BZR1), BRI1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1)/BZR2, and CESTA with putative binding sites. The classical approach using radiolabeled probes, as well as the more recent application of fluorescent probes, is described and the advantages and disadvantages of both methods are discussed.
authors.affiliation: Biotechnology of Horticultural Crops, TUM School for Life Sciences Weihenstephan, Technical University Munich, 85354, Freising, Germany.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Unterholzner
authors.affiliation: Biotechnology of Horticultural Crops, TUM School for Life Sciences Weihenstephan, Technical University Munich, 85354, Freising, Germany.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Rozhon
authors.affiliation: Biotechnology of Horticultural Crops, TUM School for Life Sciences Weihenstephan, Technical University Munich, 85354, Freising, Germany. brigitte.poppenberger@wzw.tum.de.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Poppenberger
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D018744:DNA, Plant / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D029681:Arabidopsis Proteins; C456160:BES1 protein, Arabidopsis; C454444:BZR1 protein, Arabidopsis; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D005452:Fluoresceins; D009687:Nuclear Proteins; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C000606235:gibberellin 20 oxidase 1, Arabidopsis
keywords: Competitor; Electrophoretic mobility shift assay; Fluorescence; Probe; Protein expression; Transcription factor