Assaying Cdc25 phosphatase activity

Methods Mol Biol. 2004:281:153-62. doi: 10.1385/1-59259-811-0:153.

Abstract

To determine the activity of all human Cdc25 phosphatases, two different methods are described. For assaying phosphatase activities of recombinant Cdc25 proteins produced in Escherichia coli or insect cells, a fluorimetric assay using fluorescein diphosphate (FDP) as a substrate is recommended. To analyze endogenous Cdc25 phosphatase activities of immunoprecipitates from total cellular extracts, the physiological substrate Cdk1/cyclin B1 is most sensitive.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • CDC2 Protein Kinase / metabolism*
  • Cyclin B / metabolism
  • Cyclin B1
  • Escherichia coli / metabolism
  • Fluorescein / metabolism
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • HeLa Cells
  • Humans
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Recombinant Fusion Proteins / analysis
  • S Phase
  • Spodoptera / cytology
  • Spodoptera / genetics
  • cdc25 Phosphatases / analysis*

Substances

  • CCNB1 protein, human
  • Cyclin B
  • Cyclin B1
  • Recombinant Fusion Proteins
  • Glutathione Transferase
  • Protein Kinases
  • histone H1 kinase
  • CDC2 Protein Kinase
  • cdc25 Phosphatases
  • Fluorescein