Development and optimization of high-throughput in vitro protein phosphatase screening assays.

@article{citeulike:1638356, abstract = {We describe here detailed protocols to design, optimize and validate in vitro phosphatase assays that we have utilized to conduct high-throughput screens for inhibitors of dual-specificity phosphatases: CDC25B, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3. We provide details of the critical steps that are needed to effectively miniaturize the assay into a 384-well, high-throughput format that is both reproducible and cost effective. In vitro phosphatase assays that are optimized according to these protocols should satisfy the assay performance criteria required for a robust high-throughput assay with Z-factors >0.5, and with low intra-plate, inter-plate and day-to-day variability (CV <20\%). Assuming the availability of sufficient active phosphatase enzyme and access to appropriate liquid handling automation and detection instruments, a single investigator should be able to develop a 384-well format high-throughput assay in a period of 3-4 weeks.}, address = {Department of Pharmacology, Pittsburgh Molecular Library Screening Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.}, author = {Tierno, M. B. and Johnston, P. A. and Foster, C. and Skoko, J. J. and Shinde, S. N. and Shun, T. Y. and Lazo, J. S. }, citeulike-article-id = {1638356}, doi = {http://dx.doi.org/10.1038/nprot.2007.155}, issn = {1750-2799}, journal = {Nat Protoc}, number = {5}, pages = {1134--1144}, posted-at = {2007-09-09 09:20:58}, priority = {2}, title = {Development and optimization of high-throughput in vitro protein phosphatase screening assays.}, url = {http://dx.doi.org/10.1038/nprot.2007.155}, volume = {2}, year = {2007} }

TY - JOUR ID - citeulike:1638356 L3 - citeulike-article-id:1638356 TI - Development and optimization of high-throughput in vitro protein phosphatase screening assays. JF - Nat Protoc VL - 2 IS - 5 SP - 1134 EP - 1144 AD - Department of Pharmacology, Pittsburgh Molecular Library Screening Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA. SN - 1750-2799 N2 - We describe here detailed protocols to design, optimize and validate in vitro phosphatase assays that we have utilized to conduct high-throughput screens for inhibitors of dual-specificity phosphatases: CDC25B, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3. We provide details of the critical steps that are needed to effectively miniaturize the assay into a 384-well, high-throughput format that is both reproducible and cost effective. In vitro phosphatase assays that are optimized according to these protocols should satisfy the assay performance criteria required for a robust high-throughput assay with Z-factors >0.5, and with low intra-plate, inter-plate and day-to-day variability (CV <20%). Assuming the availability of sufficient active phosphatase enzyme and access to appropriate liquid handling automation and detection instruments, a single investigator should be able to develop a 384-well format high-throughput assay in a period of 3-4 weeks. AU - Tierno, MB AU - Johnston, PA AU - Foster, C AU - Skoko, JJ AU - Shinde, SN AU - Shun, TY AU - Lazo, JS PY - 2007/// UR - http://dx.doi.org/10.1038/nprot.2007.155 ER -