A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates Journal Article
| Authors: | Wu, D.; Mand, M. R.; Veach, D. R.; Parker, L. L.; Clarkson, B.; Kron, S. J. |
| Article Title: | A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates |
| Abstract: | 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. © 2007 Elsevier Inc. All rights reserved. |
| Keywords: | controlled study; protein phosphorylation; human cell; cancer growth; sensitivity and specificity; reproducibility; imatinib; fluorescence; drug evaluation, preclinical; abelson kinase; protein tyrosine kinase; recombinant enzyme; chronic myeloid leukemia; structure-activity relationship; phosphorylation; time factors; carcinogenesis; protein tyrosine kinase inhibitor; protein kinase inhibitors; enzyme phosphorylation; enzyme regulation; quantitative analysis; peptides; bcr abl protein; biological assay; homeostasis; enzyme assay; fusion proteins, bcr-abl; cell extracts; cell lysate; phosphotyrosine; k562 cells; cross linking reagent; surface property; immunodetection; imatinib mesylate (im); kinase assay; peptide substrate; acrylic acid; cysteine derivative; thiol; hydrogel |
| Journal Title: | Analytical Biochemistry |
| Volume: | 375 |
| Issue: | 1 |
| ISSN: | 0003-2697 |
| Publisher: | Academic Press, Elsevier Inc |
| Date Published: | 2008-04-01 |
| Start Page: | 18 |
| End Page: | 26 |
| Language: | English |
| DOI: | 10.1016/j.ab.2007.12.023 |
| PUBMED: | 18194660 |
| PROVIDER: | scopus |
| PMCID: | PMC2291073 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 7" - "Export Date: 17 November 2011" - "CODEN: ANBCA" - "Source: Scopus" |
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