Abstract 5572: Analysis of changes in RTK phosphorylation using microplate-based antibody arrays

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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 effect of RTK inhibitors or modifying antibodies on RTK phosphorylation are effective tools for drug screening. The Proteome Profiler 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 pre-spotted with antibodies against each RTK. Each spot corresponds to a unique analyte. A horseradish peroxidase-conjugated 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 specific 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 Profiler 96 Human Phospho-RTK Array for analysis. NRG1- β1/HRG1- β1-dependent tyrosine phosphorylation of all four ErbB receptors was monitored simultaneously and the effects of different kinase inhibitors or modifying antibodies were determined. Proteome Profiler 96 Antibody Arrays offer the advantages of small sample and volume size requirements with as little as 5 μg of total cellular protein in a total volume of 100 μL being required, while still offering the specificity and sensitivity of sandwich immunoassays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5572.