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

2007; Nature Portfolio; Volume: 2; Issue: 5 Linguagem: Inglês

10.1038/nprot.2007.155

1754-2189

Marni Brisson Tierno, Paul A. Johnston, Caleb Foster, John Skoko, Sunita N. Shinde, Tong Ying Shun, John S. Lazo,

Protein Kinase Regulation and GTPase Signaling

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.