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JoVE Journal Biology
A Quantitative Assay to Study Protein
 

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Article DOI: 10.3791/50512-v • 06:43 min • August 31st, 2013
August 31st, 2013 •
1, 1, 2, 3, 4, 3,5
1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Program in Molecular Medicine, University of Maryland School of Medicine, 3Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, 4Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine, 5Department of Pathology and Biochemistry & Molecular Biology, University of Maryland School of Medicine

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Summary
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We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

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Quantitative Assay Protein:DNA Interactions Transcriptional Regulators Gene Expression Anti-tumor Agents DNA-binding Assays Electrophoretic Mobility Shift Assays (EMSA) Chemiluminescent Assays Chromatin Immunoprecipitation (ChIP) Multiwell-based Assays Transcription Factor Activity Radiolabeled Oligonucleotides Inhibitors Of DNA Binding Quantitative DNA-binding Enzyme-linked Immunosorbent Assay (D-ELISA) Assay RUNX2 Transcription Factor Consensus DNA-binding Sequences Biotin-labeled Oligonucleotides Cells Preparation Nuclear Protein Extraction Double Stranded Oligonucleotides Design Avidin-coated 96-well Plates Alkaline Buffer Fixation Nucleotide Blocking Buffer Incubation Primary Antibody And Secondary Antibody Incubations Horseradish Peroxidase Substrate Addition Colorimetric Reaction Development Specificity Controls
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