| Assay Method Information | |
| | SHP2 Allosteric Inhibition Assay |
| Description: | The phosphatase reaction was conducted in 384-well black polystyrene plates (Corning, Cat #3575) with flat bottom, low edge and non-binding surface at room temperature and 25 μl final volume of the following buffer condition: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.The following experiments were conducted to monitor the SHP2 inhibition by the compounds (at concentrations of 0.0003-100 μM) in this invention:Wherein, incubate 0.5 nM SHP2 with 0.5 μM peptide IRS1_pY1172 (dPEG8) pY1222 (sequence: H2N-LN (pY) IDLDLV (dPEG8) LST (pY) ASINFQK-amide) (SEQ ID NO: 1) (WO2016/203406A1). After incubation at 25° C. for 30-60 minutes, the alternative substrate DiFMUP (Invitrogen, cat #D6567) was added to the reaction and incubated at 25° C. for 30 minutes. The reaction was then carefully diluted by adding 5 μL of a 160 μM bpV (Phen) solution (Enzo Life Sciences cat #ALX-270-204). The fluorescence signal was monitored using a microplate reader (VARIOSKAN LUX, Thermo) with excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitory dose-response curve is analyzed using a standardized IC50 regression curve based on control-based normalization. |
| Affinity data for this assay | |
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