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.
Tierno
Marni Brisson
MB
Department of Pharmacology, Pittsburgh Molecular Library Screening Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Johnston
Paul A
PA
Foster
Caleb
C
Skoko
John J
JJ
Shinde
Sunita N
SN
Shun
Tong Ying
TY
Lazo
John S
JS
D015336:Molecular Probe Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000302:isolation & purification* / Q000494:pharmacology
D054852:Small Molecule Libraries; D010749:Phosphoprotein Phosphatases