Development and optimization of high-throughput in vitro protein phosphatase screening assays
| Autor: | Paul A. Johnston, Caleb Foster, Sunita Shinde, Marni Brisson Tierno, John J. Skoko, Tong Ying Shun, John S. Lazo |
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| Rok vydání: | 2007 |
| Předmět: |
Drug discovery
Phosphatase Protein Array Analysis Drug design Molecular Probe Techniques Reproducibility of Results Protein engineering Biology Proteomics General Biochemistry Genetics and Molecular Biology In vitro Glycomics Small Molecule Libraries Biochemistry Phosphoprotein Phosphatases DNA microarray |
| Zdroj: | Nature protocols. 2(5) |
| ISSN: | 1750-2799 |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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