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

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.