A Miniaturized Assay for Measuring Small Molecule Phosphorylation in the Presence of Complex Matrices

Anal Biochem. 2014 Apr 15;451:76-8. doi: 10.1016/j.ab.2013.12.010. Epub 2013 Dec 12.

Abstract

We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.

Keywords: Choline kinase; Hexokinase; Kinase; Pantothenate kinase; Phosphorylation assay; Somogyi.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Barium Compounds / chemistry
  • Chemical Precipitation
  • Escherichia coli / enzymology
  • Escherichia coli Proteins / metabolism
  • Miniaturization*
  • Pantothenic Acid / chemistry
  • Pantothenic Acid / metabolism
  • Phosphorus / analysis*
  • Phosphorus / chemistry
  • Phosphorus Radioisotopes / chemistry
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Radiometry*
  • Zinc Sulfate / chemistry

Substances

  • Barium Compounds
  • Escherichia coli Proteins
  • Phosphorus Radioisotopes
  • Pantothenic Acid
  • barium hydroxide
  • Phosphorus
  • Zinc Sulfate
  • Phosphotransferases (Alcohol Group Acceptor)
  • pantothenate kinase