| Assay Method Information | |
| | In Vitro Biochemical Assay |
| Description: | Enzyme activity assays were performed in triplicate in 384-well Greiner Bio-One black small volume microtiter plates, as previously described using recombinant human His6-tagged PTP4A1, PTP4A2, PTP4A3, PTP4A3 mutants, CDCl25B, or DUSP3 and substrate DiFMUP (12 μM) incubated at 25° C. for 25 minutes in 40 mM Tris-HCl (pH 7.0), 75 mM NaCl, 2 mM EDTA, and 4 mM DTT buffer. The assays were fully automated using an Agilent Bravo Liquid Handling Platform and miniaturized to 15 ml total volume. Dilutional reversibility assays were performed in a 100 ml total reaction volume using the same assay conditions (McQueeney et al., 2017, 2018). His6-tagged PTP4A3 (1 mg) was preincubated for 30 minutes with 0, 86, or 860 nM compound and then diluted to 10-fold. Reactions were initiated with the addition of 45 ml of substrate for a final DiFMUP concentration of 12 μM and incubated at room temperature for 25 minutes. Preincubation studies with 9p were performed by incubating the compound with PTP4A3 for 2 hours with continuous shaking, after which time substrate was added and the standard assay conditions were followed. Fluorescence data were captured on a SpectraMax M5 (San Jose, CA) and phosphatase activity was expressed as a percentage of maximal activity. |
| Affinity data for this assay | |
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