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Journal of Biomolecular Screening
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1087057106288444v1
11/6/617    most recent
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Article

Readout Technologies for Highly Miniaturized Kinase Assays Applicable to High-Throughput Screening in a 1536-Well Format

Martin Klumpp*, Andreas Boettcher, Damaris Becker, Gabriele Meder, Jutta Blank, Lukas Leder, Michael Forstner, Johannes Ottl, Lorenz M. Mayr

Novartis Institute for Biomedical Research, Basel, Switzerland.

* To whom correspondence should be addressed. E-mail: martin.klumpp{at}novartis.com.


  Abstract

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAPTM technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.

Key Words: kinase, high-throughput screening (HTS), time-resolved fluorescence resonance energy transfer (TR-FRET), fluorescence polarization (FP), chemiluminescence, assay, IMAP

First published on June 7, 2006, doi:10.1177/1087057106288444

Journal of Biomolecular Screening 2006;11:617.

A more recent version of this article appeared on September 1, 2006

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