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

Nature Protocols
Elizabeth R SharlowJohn S Lazo

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.

References

Jun 6, 2000·Journal of Biomolecular Screening·J H ZhangK R Oldenburg
Aug 26, 2000·Current Opinion in Chemical Biology·R P Hertzberg, A J Pope
May 18, 2001·Nature·P Blume-Jensen, T Hunter
May 15, 2002·Journal of Biomolecular Screening·Peter Banks, Michael Harvey
Jul 5, 2002·Journal of Biomolecular Screening·Wei HuangJ Richard Sportsman
Jul 18, 2002·Nature Reviews. Drug Discovery·Margaret A Somerville
Dec 10, 2002·Science·G ManningS Sudarsanam
Apr 16, 2003·Analytical Biochemistry·Kolbrún Kristjánsdóttir, Johannes Rudolph
Jul 8, 2003·Journal of Biomolecular Screening·Elizabeth A GaudetJ Richard Sportsman
Jul 8, 2003·Journal of Biomolecular Screening·Rommel MallariShu-Gui Huang
Jul 18, 2003·Drug Discovery Today·Christian EggelingStefan Jäger
Dec 24, 2003·Drug Discovery Today·Philip Gribbon, Andreas Sewing
Jan 9, 2004·Journal of Biomolecular Screening·Ge WuC Nicholas Hodge
Feb 6, 2004·American Journal of Physiology. Cell Physiology·A S Verkman
Apr 20, 2004·Assay and Drug Development Technologies·Tammy C Turek-EtienneRobert W Bryant
May 29, 2004·Assay and Drug Development Technologies·James R BeasleyDavid A Dunn
May 29, 2004·Assay and Drug Development Technologies·Mitsunori Koresawa, Takayoshi Okabe
May 29, 2004·Assay and Drug Development Technologies·J Richard SportsmanAnnegret Boge
Oct 21, 2004·Proceedings of the National Academy of Sciences of the United States of America·Frauke RininslandDavid Whitten
Nov 16, 2004·Science·Christopher P AustinFrancis S Collins
Jan 7, 2005·Journal of Biomolecular Screening·Heidrun RhodeAnton Horn
Mar 16, 2005·Cancer Cell·Kiranmai GumireddyE Premkumar Reddy
Mar 25, 2006·Nature Reviews. Cancer·Klaus Strebhardt, Axel Ullrich
May 9, 2006·Trends in Pharmacological Sciences·Qiming J Wang
Jun 9, 2006·Journal of Biomolecular Screening·Oliver von AhsenKarsten Parczyk
Aug 2, 2006·Molecular BioSystems·Eric Trinquet, Gérard Mathis
Sep 16, 2006·Journal of Biomolecular Screening·Sandra FoxMichael Biros
Nov 3, 2006·Nature Reviews. Drug Discovery·Solomon Nwaka, Alan Hudson
Feb 13, 2007·Current Biology : CB·Martin SteegmaierWolfgang J Rettig
Mar 16, 2007·Assay and Drug Development Technologies·Narsimha MunagalaWentao Zhang
Jun 5, 2007·Nature Protocols·Marni Brisson TiernoJohn S Lazo
Jul 3, 2007·British Journal of Pharmacology·D A Pereira, J A Williams
Jul 20, 2007·Nature Chemical Biology·James IngleseDouglas S Auld
Sep 5, 2007·Assay and Drug Development Technologies·Saloumeh KadkhodayanWayne J Fairbrother
Oct 18, 2007·Assay and Drug Development Technologies·An RykxJohan Van Lint
Jan 10, 2008·Assay and Drug Development Technologies·Paul A JohnstonAlex Y Strongin
Feb 2, 2008·Circulation Research·Metin AvkiranRobert S Haworth

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Citations

Mar 15, 2011·Analytical Chemistry·Anton IliukW Andy Tao
Oct 3, 2008·The Journal of Biological Chemistry·Elizabeth R SharlowQ Jane Wang
Feb 8, 2013·The American Journal of Tropical Medicine and Hygiene·Max GroglRichard J Sciotti
Jan 4, 2012·ACS Chemical Biology·Brandi M BaughmanThomas R Webb
Feb 14, 2012·Expert Opinion on Drug Discovery·Wendy A Lea, Anton Simeonov
Jan 30, 2016·Assay and Drug Development Technologies·Seongho Lee, Victor Sukbong Hong
Mar 3, 2010·Progress in Lipid Research·Patricia GangoitiAntonio Gómez-Muñoz
Jun 12, 2010·Angewandte Chemie·Hyun-Woo RheeJong-In Hong
May 28, 2019·Current Pharmaceutical Design·Raju DashS M Zahid Hosen
May 28, 2009·Journal of Biomolecular Screening·Michael HabigMartin Klumpp
Oct 5, 2021·Chembiochem : a European Journal of Chemical Biology·Daisuke FujiwaraIkuo Fujii

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