title: A quantitative assay to study protein:DNA interactions, discover transcriptional regulators of gene expression, and identify novel anti-tumor agents.
abstract: Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.
authors.affiliation: Greenebaum Cancer Center, University of Maryland School of Medicine.
authors.forename: Karen F
authors.initials: KF
authors.lastname: Underwood
authors.forename: Maria T
authors.initials: MT
authors.lastname: Mochin
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Brusgard
authors.forename: Moran
authors.initials: M
authors.lastname: Choe
authors.forename: Avi
authors.initials: A
authors.lastname: Gnatt
authors.forename: Antonino
authors.initials: A
authors.lastname: Passaniti
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000494:pharmacology; D003124:Colorimetry; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D050659:Core Binding Factor Alpha 1 Subunit; C493709:RUNX2 protein, human; D014157:Transcription Factors; D004247:DNA
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