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Analysis of Changes in RTK Phosphorylation Using Proteome Profiler™ 96 Antibody Arrays
David J. Finkel, Wade M. Johnson, Christopher J. Buehl, Rebecca A. Coleman, Maria T. Campos, Stefan M. Luhowskyj, Kristina M. Boldin, Steven P. Stoesz, Roberto Campos-Gonzalez, Richard A. Krzyzek, Kathryn M. Brumbaugh
Sponsored by R&D Systems, Inc.
DOI:  10.2144/000113320
Receptor-tyrosine kinases (RTKs) are transmembrane proteins that have been implicated in various cancers and are considered therapeutic targets. The phosphorylation of RTKs on tyrosine residues leads to their activation. Therefore, methods to identify the eff ect of RTK inhibitors or modifying antibodies on RTK phosphorylation are eff ective tools for drug screening. The Proteome Profi ler 96 Human Phospho-RTK Antibody Array is a unique and powerful, plate-based multiplex immunoassay that utilizes ELISA techniques to measure changes in the phosphorylation of multiple RTKs simultaneously. In this assay, antibody/antigen reactions take place on the surface of a microplate that has been prespotted with antibodies against each RTK. Each spot corresponds to a unique analyte. A horseradish peroxidaseconjugated anti-Phospho-Tyrosine detection antibody is subsequently added to each well. A chemiluminescent substrate mix and a camera imaging system* are used to determine the relative amount of analyte bound in individual spots. Experiments were performed by incubating the MDA-MB-453 breast cancer cell line with specifi c kinase inhibitors or with antibodies to ErbB receptors prior to NRG1-β1/HRG1-β1 treatment. Cells were lysed directly in 96-well plates and transferred to the Proteome Profi ler 96 Human Phospho-RTK Array for analysis. NRG1-β1/HRG1-β1-dependent tyrosine phosphorylation of all four ErbB receptors was monitored simultaneously and the eff ects of diff erent kinase inhibitors or modifying antibodies were determined. Proteome Profi ler 96 Antibody Arrays off er the advantages of small sample and volume size requirements with as little as 5 g of total cellular protein in a total volume of 100 L being required, while still off ering the specifi city and sensitivity of sandwich immunoassays.
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