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P2012
GDP Detection Using the Transcreener Technology
Presenter Karen Kleman-Leyer, BellBrook Labs, USA
Additional Authors: Mark Koeff, Tom Zielinski, Robert Lowery
Proteins that utilize purine nucleotides as substrates or cofactors, collectively referred to as the purinome, account for approximately 13% of the coding capacity of the human genome.  The Transcreener HTS assay platform relies on homogenous, fluorescent immunodetection of nucleotides, and thus provides generic detection for many enzymes in the purinome.  We previously developed Transcreener assays for ADP, AMP and GMP detection, enabling facile screening of established target families such as protein kinases and phosphodiesterases as well as emerging targets such as lipid kinases and ATPases.  In this poster, we describe the development of a homogeneous, fluorescence polarization-based TranscreenerTM assay for GDP. The binding and selectivity properties of three monoclonal antibodies raised against a purine nucleotide immunogen were characterized using a panel of far-red NDP tracers.  Differential effects of buffer components, including salts and metals, on the antibody/tracer complex were observed with the different antibodies.  Standard curves (1-1000 µM GTP) prepared with a mouse monoclonal antibody and an NDP-AlexaFluor®633 tracer demonstrated Z’ scores greater than 0.5 at <15 % GTP conversion.  This robust assay is ideal for performing HTS GTPase assays under initial rate conditions.  The Transcreener technology will facilitate the search for new inhibitors of GTPases, which control a variety of cellular events including actin cytoskeletal organization, cell growth, proliferation, differentiation, and survival.