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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
Abstract:
Many DNA binding proteins are known to regulate gene expression. When that binding is altered, a disease state can result. A common method for measuring DNA binding, namely electrophoretic mobility shift assay (EMSA) is often used but it is not amenable to rapid screening of many samples. As an alternative method, we have developed a DNA binding assay for the tumor suppressor protein p53 in a 96-well microtiter plate format using scintillation proximity assay (SPA) beads. We have shown this assay to be sensitive (as little as 0.5 ng p53 can be detected), quick (assay completed in as little as 15 min), and easily quantitated using a microtiter plate scintillation counter We also used the assay to analyze the kinetics of the DNA binding to p53. The specificity of this p53 DNA binding SPA was confirmed using competition by oligonucleotides either from the same gene or from mutated versions of this sequence. Thus, SPA is a good alternative to gel shift assays for DNA binding and may be useful for the analysis of multiple tumor cell samples or for high-throughput screens for compounds affecting DNA binding by proteins of interest.
Insights
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.
