Category: Assay Development and Screening
Phosphorylation of a protein is the most common post-translational modification used to regulate intracellular processes including cell signaling, cellular growth, and proliferation. Phospho-proteins such as STAT3 and ERK are known to be constitutively active in a variety of cancer types and are important therapeutic targets for drug development. Therefore, simple and rapid methods for screening inhibitors that block activation of these targets are highly desirable. Traditional biochemical approaches to measure kinase activity involve using a purified recombinant enzyme to phosphorylate a substrate. Screening kinase activities in cell-based assays offers the advantage of measuring a compound’s activity in a more biologically relevant context. LANCE® Ultra TR-FRET is a fast homogeneous mix-and-read assay that accurately measures phosphorylation events without the need for specially-engineered cell lines. Here we demonstrate how to optimize a LANCE Ultra TR-FRET assay to screen for activators and inhibitors of ERK phosphorylation. We also show that this highly robust assay can be used to accurately measure IC50s for three compounds that inhibit the phosphorylation of STAT3 in a variety of human cell lines. These two examples illustrate the flexibility of LANCE Ultra TR-FRET-based detection and how it can be applied across therapeutic targets.
Jeanine Hinterneder– Applications Scientist, PerkinElmer, Hopkinton, MA
Jeanine Hinterneder is an Applications Scientist in the Discovery Applications group at PerkinElmer. She received her Ph.D. from Brandeis University where her thesis work examined the mechanisms by which neurotrophic factors influence neuronal development. Jeanine has worked at PerkinElmer for the past 7 years primarily supporting the high throughput screening and microfluidic product lines. The goal of her research is to develop assays for novel targets and target classes and in vitro assays with increased biological relevance.