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Published on: February 7, 2019
Scintillation proximity assay for DNA binding by human p53
Susannah Gal1, Jeffery R Cook, Leighton Howells
1Department of Biological Sciences, SUNY-Binghamton and BioLife Solutions, Binghamton, NY 13902, USA. sgal@binghamton.edu
Biotechniques
|September 23, 2006
Summary
We developed a rapid, sensitive scintillation proximity assay (SPA) for detecting DNA binding by the tumor suppressor protein p53. This 96-well plate assay is a valuable alternative to traditional methods for high-throughput screening.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA-binding proteins regulate gene expression, and alterations in binding can lead to disease.
- Electrophoretic mobility shift assay (EMSA) is a common method for measuring DNA binding but is not suitable for high-throughput screening.
- The tumor suppressor protein p53 plays a critical role in cellular regulation.
Purpose of the Study:
- To develop a novel, high-throughput DNA-binding assay for the p53 protein.
- To establish a sensitive and rapid method for analyzing protein-DNA interactions.
- To provide an alternative to EMSA for screening multiple samples.
Main Methods:
- Developed a 96-well microtiter plate-based DNA-binding assay using scintillation proximity assay (SPA) beads for the p53 protein.
- Quantified p53 DNA binding using a microtiter plate scintillation counter.
- Analyzed the kinetics and specificity of p53 DNA binding using competition assays with varying oligonucleotides.
Main Results:
- The developed SPA assay is highly sensitive, detecting as little as 0.5 ng of p53.
- The assay is rapid, with completion in as little as 15 minutes.
- Specificity was confirmed through competition assays, demonstrating reliable detection of p53 DNA interactions.
Conclusions:
- Scintillation proximity assay (SPA) offers a sensitive, rapid, and scalable alternative to gel shift assays for studying protein-DNA interactions.
- This p53 DNA-binding SPA assay is suitable for analyzing numerous tumor cell samples.
- The assay can be utilized for high-throughput screening of compounds that modulate protein-DNA binding.
