| Assay Method Information | |
| | PTPN2 Biochemical Assay |
| Description: | Test compounds were dissolved in DMSO, and 10-point serial 3-fold dilution series in DMSO were prepared in Echo Qualified 384-well Polypropylene Microplates (Labcyte, San Jose, CA) (top concentration 50 M). Assay plates (384-well low volume black plates; Corning #3820, Corning, NY) were prepared by dispensing 50 nL of test compound, and DMSO (for high and low controls) by ECHO acoustic dispenser (Labcyte, San Jose CA). This was followed by addition of 5 μL of 0.2 nM human PTPN2 (1-387) solution (prepared in the assay buffer, 50 mM Tris pH 7.4, 150 mM NaCl, 0.01% Tween20, 0.5 mM dithiothreitol) to all wells except the low control. 5 μl of assay buffer was added into the low control, then the plate was incubated for 30 minutes at room temperature. Subsequently, 5 μl of 100 M DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) solution (prepared in assay buffer from 10 mM stock in DMSO) was added to the assay plate and incubated for 1 hour at room temperature. For detection, assay plates were read on SpectraMax Microplate Reader (Molecular Devices), with Excitation wavelength=360 nm and Emission wavelength=460 nm. |
| Affinity data for this assay | |
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