A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

Ding Wu, Michael R. Mand, Darren R. Veach, Laurie L. Parker, Bayard Clarkson, Stephen J. Kron

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)18-26
Number of pages9
JournalAnalytical Biochemistry
Issue number1
StatePublished - Apr 1 2008
Externally publishedYes


  • Imatinib mesylate (IM)
  • Kinase assay
  • Peptide substrate
  • Protein tyrosine kinase

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