| Assay Method Information | |
| | Assay of Full-Length SHP2 Phosphatase Activity Assay |
| Description: | 1. Reagents and MaterialsHuman full-length SHP2 recombinant protein: BPS Bioscience, Cat #79018;SHP2 substrate DiFMUP (1 mM): BPS Bioscience, Cat #79769;SHP2 activating peptide (100 μM): BPS Bioscience, Cat #79319-2;DTT: Merck, Cat #DTT-RO;384-well plate: Corning, Cat #3575;96-well plate: Thermo Fisher Scientific, Cat #249952;Instrument: EnVision 2104, PerkinElmer.2. Preparation of Reaction SolutionsThe test compound was dissolved in DMSO and diluted with DMSO to 100.0 μM, and the compound was further 3-fold diluted with DMSO to: 100.00, 33.33, 11.11, 3.70, 1.23, 0.41, 0.14 and 0.05 μM. Then 4 μL of the compound at different dilution concentrations was added to 96 μL of an enzymatic reaction buffer to prepare a 4× test compound, wherein DMSO was at the concentration of 4% (DMSO was at the final concentration of 1%).Preparation of 1× enzymatic reaction buffer: the 5× reaction buffer (250 mM HEPES, 500 mM NaCl, 2.5 mM EDTA, 0.005% Brij-35 and 0.01% BSA, pH 7.2) was diluted 5-fold with deionized water, and then DTT was added thereto so that the 1× enzymatic reaction buffer contained 5 mM DTT.Preparation of 4× mixed solution of SHP2 enzyme/activating peptide: the SHP2 enzyme (75.5 nM) and activating peptide (100 μM) were diluted with the enzymatic reaction buffer to prepare a 4× mixed solution of SHP2 enzyme/activating peptide (0.12 nM SHP2 and 2 μM activating peptide), so that the SHP2 enzyme and activating peptide were at the final concentrations of 0.03 nM and 0.5 μM in the enzymatic reaction system, respectively.Preparation of 2×DiFMUP substrate: 1 mM DiFMUP was diluted 100-fold with the enzymatic reaction buffer to prepare a 2× substrate (10 μM), so that the substrate DiFMUP was at the final concentration of 5 μM in the enzymatic reaction system. |
| Affinity data for this assay | |
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