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Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries

Nature Protocols volume 3, pages 1350–1363 (2008)Cite this article

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 ∼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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Figure 1: Flowchart of high-throughput screening (HTS) stages of development.
Figure 2: A UPDDI-specific screening paradigm to confirm the activity of low-molecular-weight inhibitors identified in IMAP FP/TR-FRET HTS assays.
Figure 3: Assay plate setup for IMAP FP and time-resolved fluorescence resonance energy transfer (TR-FRET) assay formats.
Figure 4: Typical MAX and MIN signals from PKD IMAP FP and PLK1 TR-FRET HTS assays.

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Acknowledgements

This work was supported by the National Institutes of Health grants R03 DA24898-01, U54 MH074411-03 and X01 MH077611-01. We thank Dr. Paul A. Johnston for his technical advice with respect to HTS assay development and implementation.

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  1. Department of Pharmacology and Chemical Biology, Drug Discovery Institute, University of Pittsburgh, Pittsburgh, 15260, Pennsylvania, USA

    Elizabeth R Sharlow, Stephanie Leimgruber, Archibong Yellow-Duke, Rebecca Barrett, Qiming Jane Wang & John S Lazo

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Correspondence to Elizabeth R Sharlow.

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Sharlow, E., Leimgruber, S., Yellow-Duke, A. et al. Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries. Nat Protoc 3, 1350–1363 (2008). https://doi.org/10.1038/nprot.2008.111

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