| Assay Method Information | |
| | Inhibition of SHP2 Phosphatase Activity Assay |
| Description: | Inhibition of SHP2 phosphatase activity was evaluated using human recombinant 6His-Nterm (SEQ ID NO: 1) SHP2 full-length (Thr2-Arg593, R&D system), NsCs bi-phosphorylated activating peptide (NH2-LN(pY)AQLWHA(PEG8)LTI(pY)ATIRRF-Amide.acetate (SEQ ID NO: 2), Thermofischer Scientific) and DiFMUP as surrogate phosphatase substrate (Molecular Probes). All reactions were performed at room temperature in 384-well black polystyrene non-binding plate (Corning) with the assay buffer (50 mM HEPES-NaOH pH 7.2, 100 mM NaCl, 0.5 mM EDTA, 0.001% Brij35 including 0.02% BSA and 1 mM DTT added extemporaneously). SHP2 was pre-incubated at least 10 min with the activating peptide (0.2 nM and 100 nM, respectively). 10 μL of this mixture was added to the assay plate, that already contained 15 nL of the compound (1000-0.05 nM). After 30 min pre-incubation, the enzymatic reaction was initiated by the addition of 5 μL DiFMUP in assay buffer without BSA (20 μM). The reaction was stopped by the addition of 15 μL sodium pervanadate solution (100 μM) after 45 min incubation. The fluorescence signal was measured at excitation and emission wavelengths of 350 nm and 450 nm, respectively, using a Pherastar instrument (BMG-LabTech). |
| Affinity data for this assay | |
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