| Assay Method Information | |
| | Test of Inhibitory Activity Assay |
| Description: | (1) Preparing 1×Reaction Buffer according to the BPS Biosciences' Instructions for Use of the SHP2 enzyme. (2) Preparing a compound concentration gradient: Test the test compound at 10 μM under 3× dilution for 10 concentrations, diluting to a 100% dimethyl sulfoxide solution at a 100× final concentration in a 384-well plate, and diluting the compound with Precision 4× for 10 concentrations. Transferring 250 nL of the compound at the 100× final concentration to a target plate OptiPlate-384F using a dispenser Echo 550. Adding 250 nL of dimethyl sulfoxide to a positive control and 250 nL of 1 mM SHP099 to a negative control. (3) Preparing the activation peptide solution at a 5× final concentration with the 1×Reaction Buffer, adding 5 μL of the solution to a reaction plate, respectively, and centrifuging at 1000 rpm for 1 min. (4) Preparing an enzyme solution at a 2.5× final concentration with the 1×Reaction Buffer, adding 10 μL of the solution to the reaction plate, respectively, centrifuging at 1000 rpm for 1 min, and incubating at room temperature for 60 min. (5) Preparing a substrate peptide solution at a 2.5× final concentration with the 1×Reaction Buffer, centrifuging at 1000 rpm for 1 min, and incubating at room temperature for 30 min. (6) Adding 30 μL of stop test solution to stop the reaction, centrifuging at 1000 rpm for 60 see, and mixing well by shaking. (7) Reading the conversion rate with a Caliper EZ Reader. (7) Data analysis:%Inhibition=Conversion%_max-Conversion%_sampleConversion%_max-Conversion%_min×100Wherein: Conversion %/_sample is the reading of a conversion rate of a sample; Conversion %_min: mean of negative control wells, which represents the reading of a conversion rate in absence of enzyme activity wells; Conversion %_max: mean of a ratio of positive control wells, which represents the reading of a conversion rate in absence of compound inhibition wells. |
| Affinity data for this assay | |
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