A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates Academic Article uri icon


MeSH Major

  • Biological Assay
  • Fusion Proteins, bcr-abl
  • Hydrogel


  • Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.

publication date

  • April 2008



  • Academic Article



  • eng

PubMed Central ID

  • PMC2291073

Digital Object Identifier (DOI)

  • 10.1016/j.ab.2007.12.023

PubMed ID

  • 18194660

Additional Document Info

start page

  • 18

end page

  • 26


  • 375


  • 1