| Assay Method Information | |
| | SHP2 Phosphatase Assay |
| Description: | IC50 values were determined at room temperature in 384-well black polystyrene plate, using a final reaction volume of 15 μl and the following assay buffer conditions: 60 mM Hepes (pH=7.2), 75 mM NaCl, 75 mM KCl, and 1 mM EDTA, 0.05% P-20, 5 mM dithiothreitol (DTT). Full length SHP2 enzyme (diluted to 0.1 nM in reaction buffer) were co-incubated with 1 uM IRS-1 peptide and 0.01 nM to 10 μM compounds of the disclosure for 60 min. The surrogate substrate DiFMUP (5 μL, 100 LIM) was added, and incubated at rt for 60 min. The reaction was then quenched by the addition of 5 μL of a 40 μM solution of bpV(Phen). The fluorescence signal was monitored using a microplate reader (Envision, Perkin-Elmer) using excitation and emission wavelengths of 360 nm and 450 nm, respectively. The inhibitor dose-response curves were analyzed using normalized IC50 regression curve fitting with control-based normalization. |
| Affinity data for this assay | |
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