| Assay Method Information | |
| | PhosphoSens Assay |
| Description: | A PhosphoSens kinase assay was performed as described by the vendor (AssayQuant Technologies, Marlborough, MA). Briefly, 1000x solutions of compounds were prepared in DMSO via serial dilution of the 10 mM DMSO stocks using 3-fold intervals in a 384-well reagent plate. 50 nL of the compound dilution series was then added to the corresponding wells of a 384-well assay plate. 40 mL of 1.25x substrate (AQT0264) in 1x assay buffer (50 mM HEPES pH 7.5, 500 uM EGTA, 10 nM MgCl2, 0.01% Brij-35, 1% Glycerol, 1 mM DTT, and 0.2 mg/mL BSA) was transferred to each well of the assay plate to achieve a final substrate concentration of 20 uM. Finally, 10 mL of 5xPTPN2 enzyme stock was added to each well of the assay plate for a final enzyme concentration of 150 pM. Reaction progress curves were collected by sampling fluorescence intensity at the excitation wavelength 360 nm (Quex360) and emission wavelength 480 nm (lambdaem480) every 71 seconds for one hour using a Synergy H4 plate reader (BioTek Instruments/Agilent Technologies, Winooski, VT) at room temperature. |
| Affinity data for this assay | |
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