Abstract
The proteomics analysis of protein kinases and other cell-signaling proteins in tumor samples by traditional two-dimensional (2-D) gel electrophoresis is complicated by the low abundance of these regulatory proteins relative to metabolic enzymes and structural proteins. We present an antibody-based method called Kinetworks™ that relies on sodium dodecyl sulfate (SDS)-polyacrylamide minigel electrophoresis and multilane immunoblotters to permit the specific and quantitative detection of 45 or more protein kinases or other signal transduction proteins at once. The technique can also permit the resolution of these proteins based on differences in their phosphorylation state and other forms of covalent modification. Kinetworks™ profiling of protein kinases in solid human tumors and cell lines can reveal profound differences in their expression and phosphorylation states, which can serve for the identification of cancer diagnostic markers and therapeutic targets for drug discovery.
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O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Bradford, M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–684.
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© 2003 Humana Press Inc.
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Pelech, S., Sutter, C., Zhang, H. (2003). Kinetworks™ Protein Kinase Multiblot Analysis. In: Terrian, D.M. (eds) Cancer Cell Signaling. Methods in Molecular Biology™, vol 218. Humana Press. https://doi.org/10.1385/1-59259-356-9:99
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DOI: https://doi.org/10.1385/1-59259-356-9:99
Publisher Name: Humana Press
Print ISBN: 978-1-58829-075-5
Online ISBN: 978-1-59259-356-9
eBook Packages: Springer Protocols