| Assay Method Information | |
| | Wip1 Length and Wip1(2-420) Phosphatase Enzyme FDP Assays |
| Description: | Test compounds (252 mM stock in DMSO) were diluted 3 folds in series in DMSO and 1.2 μl per well were added into 384-well polypropylene black plates (Nunc). Twelve μl per well of Assay Buffer (100 mM NaCl, 50 mM MOPS, pH 7.5, 0.8 mM CHAPS, 0.1 mM EGTA, 30 mM MgCl2, 0.3 mM TCEP, 0.2 mg/mL BSA) was added followed by 12 ml per well of 50.4 nM recombinant His-tag Wip1 full length protein (amino acids #1-605) or recombinant His-tag Wip1(2-420) protein (amino acids #2-420) in Assay Buffer and the plate was incubated at RT (22° C.) for 5 min. Ten mM of fluorescein diphosphate (FDP) in DMSO was diluted to 40 mM in Assay Buffer and 5 ml per well was added to the plate and incubated at RT (22° C.) for 2 hours. Four ml per well of 64.1 mM EDTA in water were added to stop the enzyme reaction. The plate was centrifuged at 1,200 rpm (Eppendorf 5810R Plate centrifuge) for 1 minute and fluorescence intensity (Ex485/Em535) was measured on Perkin Elmer Victor Reader. EC50 values were calculated using either Prism (GraphPad) or ActivityBase software (IDBS). |
| Affinity data for this assay | |
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