Scintillation proximity assay for DNA binding by human p53

Susannah Gal, Jeffery R. Cook, Leighton Howells

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)303-310
Number of pages8
JournalBioTechniques
Volume41
Issue number3
DOIs
StatePublished - Sep 1 2006

Fingerprint

Scintillation
Assays
DNA
DNA-Binding Proteins
Tumor Suppressor Protein p53
Scintillation Counting
Electrophoretic Mobility Shift Assay
Oligonucleotides
Gels
Electrophoretic mobility
Scintillation counters
Gene Expression
Gene expression
Tumors
Screening
Genes
Neoplasms
Cells
Throughput
Kinetics

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Gal, Susannah ; Cook, Jeffery R. ; Howells, Leighton. / Scintillation proximity assay for DNA binding by human p53. In: BioTechniques. 2006 ; Vol. 41, No. 3. pp. 303-310.
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Scintillation proximity assay for DNA binding by human p53. / Gal, Susannah; Cook, Jeffery R.; Howells, Leighton.

In: BioTechniques, Vol. 41, No. 3, 01.09.2006, p. 303-310.

Research output: Contribution to journalArticle

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