期刊
NATURE PROTOCOLS
卷 2, 期 5, 页码 1134-1144出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.155
关键词
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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 40.5, 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.
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