Establishment of a cell-free system to study the activation of Chk2

Methods Mol Biol. 2004:280:165-74. doi: 10.1385/1-59259-788-2:165.

Abstract

The checkpoint kinase Chk2 is activated in response to DNA damage through pathways requiring protein kinases ATM and/or ATR. The means by which Chk2 is activated by these kinases still remains to be addressed. Here we describe a cell-free system to study the activation of Chk2. Chk2 produced by a wheat germ extract in vitro transcription/translation system is inactive and can be activated by incubating with a rabbit reticulocyte lysate. This method will be useful for identification of cofactors required for activation of Chk2.

Publication types

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

MeSH terms

  • Animals
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins
  • Cell-Free System
  • Checkpoint Kinase 2
  • DNA Damage
  • DNA-Binding Proteins
  • Genetic Vectors
  • Humans
  • Immunoblotting
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Biosynthesis
  • Protein Serine-Threonine Kinases / metabolism*
  • Rabbits
  • Reticulocytes / metabolism
  • Transcription, Genetic
  • Triticum / metabolism
  • Tumor Suppressor Proteins

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Tumor Suppressor Proteins
  • Checkpoint Kinase 2
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK2 protein, human
  • Protein Serine-Threonine Kinases