Scintillation proximity assay for DNA binding by human p53
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 alte...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2006-09-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/000112222 |
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| _version_ | 1850152705779040256 |
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| author | Susannah Gal Jeffery R. Cook Leighton Howells |
| author_facet | Susannah Gal Jeffery R. Cook Leighton Howells |
| author_sort | Susannah Gal |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-3e1de05120af4d64ae491bafdf9c2c14 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2006-09-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-3e1de05120af4d64ae491bafdf9c2c142025-08-20T02:25:54ZengTaylor & Francis GroupBioTechniques0736-62051940-98182006-09-0141330330810.2144/000112222Scintillation proximity assay for DNA binding by human p53Susannah Gal0Jeffery R. Cook1Leighton Howells21SUNY-Binghamton and BioLife Solutions, Binghamton, NY2Johnson & Johnson Pharmaceutical Research and Development, Raritan, NJ3GE Healthcare, Piscataway, NJ, USAMany 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.https://www.future-science.com/doi/10.2144/000112222 |
| spellingShingle | Susannah Gal Jeffery R. Cook Leighton Howells Scintillation proximity assay for DNA binding by human p53 BioTechniques |
| title | Scintillation proximity assay for DNA binding by human p53 |
| title_full | Scintillation proximity assay for DNA binding by human p53 |
| title_fullStr | Scintillation proximity assay for DNA binding by human p53 |
| title_full_unstemmed | Scintillation proximity assay for DNA binding by human p53 |
| title_short | Scintillation proximity assay for DNA binding by human p53 |
| title_sort | scintillation proximity assay for dna binding by human p53 |
| url | https://www.future-science.com/doi/10.2144/000112222 |
| work_keys_str_mv | AT susannahgal scintillationproximityassayfordnabindingbyhumanp53 AT jefferyrcook scintillationproximityassayfordnabindingbyhumanp53 AT leightonhowells scintillationproximityassayfordnabindingbyhumanp53 |