A51

Multiplex analysis of Akt/mTOR and JAK/STAT signaling pathways

Nisar Pampori, Laura Schaefer, Paula Eason,Robert Umek, Jacob Wohlstadter

Meso Scale Discovery, Gaithersburg, MD, USA

Phosphoproteins play a major role in cellular signaling throughout development and in adult organisms. The importance of these proteins in normal and disease states makes them ideal candidates for drug development both as therapeutic targets and as biomarkers of cellular behavior. The experimental characterisation of phosphoproteins is made more efficient through the use of multiplex assays in microtiter plate formats. Here we present a number of multiplex, microtiter plate-based assays that facilitate the study of the Akt/mTOR pathway and the JAK/STAT pathway. The assays are very sensitive, affording detection from sub-microgram quantities of total protein and thus consistent with 384-well tissue culture applications. The results obtained with these multiplex assays are in agreement with conventional analysis by western blot with phospho-specific antibodies. Assay time is rapid and thus consistent with medium to high throughput workflows. The assays have been shown to work with a wide variety of cell types. Results from HeLa, Jurkat, HEK 293, MCF-7 and human T cells are shown here.