A method for quantification of nucleotides and nucleotide analogues in thymidine kinase assays using lanthanum phosphate coprecipitation

S. T. Gammon, M. Bernstein, D. P. Schuster, D. Piwnica-Worms

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Current methodologies for quantifying radiolabeled nucleoside monophosphates and nucleoside analogues result in high retention of unphosphorylated guanosine nucleosides in the case of lanthanum chloride precipitation or inconsistent retention of nucleotides in the case of DEAE cellulose filter papers. This study describes an innovative method for quantifying thymidine kinase (TK) activity that is compatible with both purine and pyrimidine nucleoside analogues by using lanthanum phosphate coprecipitation at pH 4.0. This methodology maintains quantitative precipitation of nucleoside monophosphates and yields minimal background binding from a variety of nucleoside analogues. In addition, use of PCR thermocyclers enhances the temporal precision of TK assays. This method was shown to be useful for assaying TK activity in a broad range of biochemically relevant systems, including purified enzymes, stable cell lines, and virally infected cells. Use of this methodology should aid researchers in the evaluation of novel nucleoside analogues and TK enzymes while decreasing radioactive waste, minimizing assay time, increasing accuracy, and enhancing dynamic range.

Original languageEnglish (US)
Pages (from-to)80-86
Number of pages7
JournalAnalytical Biochemistry
Volume369
Issue number1
DOIs
StatePublished - Oct 1 2007

Keywords

  • Lanthanum phosphate
  • Nucleoside
  • Nucleotide
  • Thymidine kinase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'A method for quantification of nucleotides and nucleotide analogues in thymidine kinase assays using lanthanum phosphate coprecipitation'. Together they form a unique fingerprint.

Cite this