This Article Full Text (PDF) All Versions of this Article: 1087057106288444v1 11/6/617    most recent References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Klumpp, M. Articles by Mayr, L. M. Search for Related Content PubMed PubMed Citation Articles by Klumpp, M. Articles by Mayr, L. M. Social Bookmarking                   What's this?

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

Novartis Institute for Biomedical Research, Basel, Switzerland

Andreas Boettcher

Novartis Institute for Biomedical Research, Basel, Switzerland

Damaris Becker

Novartis Institute for Biomedical Research, Basel, Switzerland

Gabriele Meder

Novartis Institute for Biomedical Research, Basel, Switzerland

Jutta Blank

Novartis Institute for Biomedical Research, Basel, Switzerland

Lukas Leder

Novartis Institute for Biomedical Research, Basel, Switzerland

Michael Forstner

Novartis Institute for Biomedical Research, Basel, Switzerland

Johannes Ottl

Novartis Institute for Biomedical Research, Basel, Switzerland

Lorenz M. Mayr

Novartis Institute for Biomedical Research, Basel, Switzerland

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 (IMAP^TM 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

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. Graf, M. Klumpp, M. Habig, P. Rovina, A. Billich, T. Baumruker, B. Oberhauser, and F. Bornancin Targeting Ceramide Metabolism with a Potent and Specific Ceramide Kinase Inhibitor Mol. Pharmacol., October 1, 2008; 74(4): 925 - 932. [Abstract] [Full Text] [PDF]

				L. M. Mayr and P. Fuerst The Future of High-Throughput Screening J Biomol Screen, July 1, 2008; 13(6): 443 - 448. [Abstract] [PDF]

				T. Schroter, D. Minond, A. Weiser, C. Dao, J. Habel, T. Spicer, P. Chase, P. Baillargeon, L. Scampavia, S. Schurer, et al. Comparison of Miniaturized Time-Resolved Fluorescence Resonance Energy Transfer and Enzyme-Coupled Luciferase High-Throughput Screening Assays to Discover Inhibitors of Rho-Kinase II (ROCK-II) J Biomol Screen, January 1, 2008; 13(1): 17 - 28. [Abstract] [PDF]