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P2039Comparison of a Cellular ELISA & AlphaScreen SureFire™ for Evaluation of Ligand Stimulated ERK Phosphorylation in Human Umbilical Vein Endothelial Cells
Presenter Xiu-juan Yuan, Eli Lilly & Company, USA
Additional Authors: Xiu-Juan Yuan, Jeffrey K. Smallwood, Jason R. Manro, Patrick R. Connor and Laura J. Bloem
The mitogen activated protein kinases (MAPK) are a highly conserved family of signal transduction molecules that transmit extra cellular signals from the membrane to the nucleus. One of the major members of the MAPK family of signaling molecules is the extracellular signal-regulated kinase (ERK). The location of this kinase downstream of the receptor makes measuring the phosphorylation ideal for evaluating pathway activation/inhibition in the presence of small molecules. SureFire™ is a new homogenous assay format for measuring ERK phosphorylation in cells. In this assay system, Activated ERK is captured using a biotinylated antibody to ERK and a second antibody recognizing phosphorylated ERK. Modified proteins binding both antibodies are detected using an AlphaScreen system (Perkin Elmer) containing streptavidin coated donor beads and Protein A coated acceptor beads. A growth factor stimulated assay using the SureFire™ system to measure pERK was developed and validated. A set of known growth factor inhibitors was tested for IC50 using the validated pERK assay. Values generated using the AlphaScreen format were significantly correlated to those generated using a standard fixed cellular ELISA format for measuring pERK.