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Automation of a Novel Cell-Based ELISA for Cell Signaling Pathway AnalysisDownload
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February 08, 2012
Authors: Wendy Goodrich and Peter Banks, BioTek Instruments, Inc., Winooski, Vermont, USA; Ron Osmond and Antony Sheehan, TGR Biosciences, Adelaide, Australia
Monitoring and quantifying cell signaling pathways is critical for understanding the behavior of cell processes and many disease states. Protein kinases involved in these cellular cascades play many diverse biological roles including normal growth and development; their aberrant behavior is linked to a number of infirm states including cancer. A novel ELISA assay has been developed for detection of cell based signal transduction events. In addition to offering simplicity, speed, and flexibility, the technology is a highly sensitive technique that lends itself to automation at multiple levels including seeding cells in microplates; cell media exchanges; serial dilution and transfer steps; assay reagent additions; microplate washing; and, signal detection. Assay performance studies were conducted using lysate kit controls to verify optimal automation performance for S/B, Z’, and intra- and inter-assay precision. A ‘one well one day’ cell based assay protocol was developed and used for pharmacology assessment for agonist/antagonist analysis of the PI-3/AKT/mTOR signaling pathway. Procedures demonstrated here are well suited for validating automation of model system protocol development.