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

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

DOI:
10.3791/50512-v

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06:43 min

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August 31, 2013

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, , , , ,
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

Chapters

  • 00:05Title
  • 01:49Cell Culture and Nuclear Protein Isolation
  • 02:46Preparation of 96-well Plates by Incubation with Nuclear Extracts
  • 03:58Measurement of RUNX2 DNA Binding
  • 05:36Results: Inhibition of RUNX2 DNA Binding
  • 06:22Conclusion

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.

Tags

Keywords: Protein:DNA InteractionsTranscriptional RegulatorsGene ExpressionAnti-tumor AgentsDNA-binding AssayEMSAChIPD-ELISARUNX2Protein-DNA InteractionQuantitative AssayDrug Screening

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