Detection of protein tyrosine kinase activity using a high-capacity streptavidin-coated membrane and optimized biotinylated peptide substrates.
A protein tyrosine kinase (PTK) assay system is described that uses a series of optimized biotinylated peptide substrates in conjunction with a streptavidin-coated matrix (SAM(2)) biotin capture membrane. The SAM(2) biotin capture membrane provides low backgrounds and high linear binding capacity (up to approximately 3.6 nmol of biotinylated PTK peptide/cm(2)), resulting in high signal-to-noise ratios and greater reproducibility. Capture of the phosphorylated peptide substrates onto the SAM(2) membrane is rapid and occurs independent of the amino acid sequence of the peptide, thereby overcoming difficulties commonly encountered with other methodologies. Two broad-specificity biotinylated PTK peptide substrates were identified with optimum kinetic properties, allowing members from eight distinct classes of enzymes, including transmembrane (epidermal growth factor receptor (EGFR), fibroblast growth factor receptor, insulin receptor, and platelet-derived growth factor receptor) and cytoplasmic (p43(abl), p56(lck), p60(src), and p93(fes)) PTKs, to be analyzed. A third biotinylated peptide substrate, shown to be highly selective for the EGFR, was used to illustrate the versatility of this system for both broad specificity and highly selective detection of PTK activity. The ability to accurately detect activity under optimum conditions and with crude cell extract samples, including kinetic analysis and with enzyme detection limits in the low femtomole range, supports the utility of this assay system for studying PTK enzymes.
Signal Transduction, Promega Corporation, Madison, WI 53711, USA. Eschaefer2@aol.com
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Schaefer
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Guimond
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin; D016466:CHO Cells; D002623:Chemistry Techniques, Analytical / Q000379:methods / Q000706:statistics & numerical data; D006224:Cricetinae; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008567:Membranes, Artificial; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013379:Substrate Specificity
D008567:Membranes, Artificial; D009842:Oligopeptides; D010455:Peptides; D001710:Biotin; D019809:Streptavidin; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases