A Homogeneous Assay to Quantify Endogenous AKT Phosphorylation in Human Umbilical Endothelial Cells

Brad Larson, Peter Banks, Stephanie Nickles, George Klarmann, Sylvie Crawford

2006

BioTek Instruments, Vermont, USA. Lonza, Maryland, USA. Cisbio US, Massachusetts, USA.

AKT is a serine/threonine protein kinase that plays a role in key cellular processes. These include the cell cycle, metabolism, and angiogenesis. Activated AKT has been implicated in the proliferation and survival of cells, leading to tumor development. Because of these functions, AKT has become a popular target for drug discovery campaigns, due to the fact that AKT inhibitors may help to treat a number of cancers. Here we demonstrate an automated homogeneous assay to probe AKT phosphorylation at its serine 473 residue using endogenous levels of kinase expression within human primary HUVEC cells. Validation data demonstrate that the combination of assay, cells, and instrumentation are sensitive enough to detect endogenous phosphorylation of this important drug target.

HTRF microplate readers