Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries |
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Authors: | Sharlow Elizabeth R Leimgruber Stephanie Yellow-Duke Archibong Barrett Rebecca Wang Qiming Jane Lazo John S |
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Institution: | Drug Discovery Institute, Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA. ers@pitt.edu |
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Abstract: | This protocol describes assay development, validation and implementation of automated immobilized metal affinity for phosphochemicals (IMAP)-based fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening (HTS) assays for identification of low-molecular-weight kinase inhibitors. Both procedures are performed in miniaturized kinase reaction volumes and involve the stepwise addition of test or control compounds, enzyme and substrate/ATP. Kinase reactions are stopped by subsequent addition of IMAP-binding buffer. Assay attributes of the IMAP FP and TR-FRET methodologies are described. HTS assays developed using these procedures should result in Z-factors and low assay variability necessary for robust HTS assays. Providing that the required reagents and equipment are available, one scientist should be able to develop a 384-well, miniaturized HTS assay in approximately 6-8 weeks. Specific automated HTS assay conditions will determine the number of assay plates processed in a screening session, but two scientists should expect to process between 100 and 150 assay plates in one 8-h screening day. |
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