Parallel, high throughput quantitation of kinase activity in cell extracts has applications in biomedical research and medical diagnostics. Kinetic analysis of signal transduction pathways can provide insight into cellular processes such as mitosis and differentiation. In addition, many disease states are associated with upregulated levels of cellular signaling molecules. However, accurate measurements of kinase activity directly from cell extracts is complicated by the complex nature of the extracts, as they contain multiple kinases as well as phosphatases.
We have developed a protein array based assay to quantify the activity of multiple tyrosine kinases in cell extracts. Substrates for different kinases are spotted onto a surface and covalently linked into a polyacrylamide hydrogel. This hydrogel provides a porous three dimensional support for the kinase substrates and maintains the substrates in a hydrated environment. Cell extract is prepared and incubated over the array. Phosphorylation of the substrates can be detected using fluorescently-labeled antiphosphotyrosine antibodies and scanning the array for fluorescence.
Schematic for copolymerization of acryl-labeled proteins into a polyacrylamide gel
Glutathione-S-Transferase Green Fluorescent Protein (GST-GFP) fusion protein arrayed on a glass microscope slide
We are currently developing direct methods to detect phosphorylation of the immobilized substrates and applying this assay to high throughput analysis of signal transduction networks and to screening of chemical kinase inhibitors.
Copyright 2005 The Board of Regents of the University of Wisconsin System
Date last modified:
12-Sep-2005
Date created: 8-Sep-2005
Content by: palecek@engr.wisc.edu
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