Recursos Técnicos - Posters Científicos
Optimization and Validation of a high-throughput homogeneous Time-Resolved Fluorescence (HTRF®) assay for inhibitors to Class IA Phosphoinositide 3-kinase isoform activityDescargar
April 29, 2009
Authors: Brad Larson, Peter Banks, Peter Brescia, Paul Held, BioTek Instruments, Inc; Andrew Plater, Millipore UK Ltd.
Phosphoinositide 3-kinases (PI 3-kinases) constitute a family of related enzymes that phosphorylate the 3 position hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns). PI 3-kinases have been linked to an extraordinarily diverse group of cellular functions including cell growth, proliferation, differentiation, motility, survival, and intracellular trafficking. Class IA PI 3-kinases are heterodimeric molecules composed of one of five different regulatory subunits (p85α, p55α, p50α, p85β or p55γ) and one of three different catalytic subunits (p110α, p110β, or p110δ). They are attractive small molecule drug targets as aberrant activity is linked to numerous diseases including cancer, inflammation and diabetes. Yet development of assays to screen compounds against these targets has been problematic due to the difficulty of generating antibodies that bind specifically to only one of the phosphorylation states of PtdIns (e.g., 4,5-PtdIns [PIP-2], 3,4,5-PtdIns [PIP-3], etc.). To combat this problem, Millipore has developed a PI3-Kinase HTRF® Assay that makes use of the specific, high affinity binding of the GRP1 pleckstrin homology (PH) domain to PIP3, the product of a Class 1A or 1B PI3-Kinase acting on its physiological substrate PIP2. During the detection phase of the assay, a complex is generated between the GSTtagged PH domain and biotinylated short chain PIP3, forming the basis for the FRET architecture. By combining this assay with BioTek’s Synergy™ 4 Hybrid Multi-Mode Microplate Reader, which has been previously validated to read all HTRF assays, researchers gain a way to generate high-throughput screening and pharmacology data for this important target class.