| Activity Measurement of TNF-α Converting Enzyme (TACE) in 3456-Well Nanoplate Format |
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Presentation: P03024 Session: Robotic Screening & Automation Technologies - Poster SessionMichael Bohanon, Molly Hood, Chiu-Fai Man, Piet de Dreu, Scott Wise, Tim Braden and Adam Johnson, Pfizer, Inc. Presenting Author: Michael Bohanon, Pfizer - USA Excess activity of the Zn2+ dependent protease TACE results in a pathological elevation of the cytokine TNF-α that has been implicated in disease progression of rheumatoid arthritis (RA) and other conditions with an inflammatory component. An internally quenched Cy3B/Cy5Q peptide substrate and functionally purified naturally expressed human TACE from cultured MonoMac-6 cells were used to develop and implement an Ultra-High Throughput Screen (uHTS) in 3456-well nanoplates. The hardware was all obtained from Aurora Discovery Inc. and consisted of the following. Compound spotting (10 nL) into the 3456-well nanoplate was performed with a Piezo-electric Sample Distribution Robot (PSDR). The 600 nL enzyme and substrate reagent additions were accomplished with a Flying Reagent Dispenser™ (FRD). Pseudo-kinetic reads (difference between pre/post incubation) were obtained using the Topology Compensating Plate Reader™ (tcPR). Development and adapting the TACE assay into a low volume high-density format will be discussed. |