| Assay Method Information | |
| | Inhibitory Activities of Compounds on mTOR Kinase |
| Description: | (1) Preparation of 1× kinase buffer50 mM HEPES, pH 7.51 mM EGTA0.01% Tween-20(2) Preparation of test compound1) The compound was dissolved in DMSO to yield a stock solution. Before the assay, the stock solution of the compound was diluted with DMSO to yield a 100× solution at a concentration that is 100 times a target concentration for assay. If the target concentration was M, the stock solution should be diluted to yield a 1 mM solution at this step.2) 100 μL of DMSO was added, respectively, into two blank wells in the same 96 well plate, corresponding to the total reaction control without compound and the blank control without enzyme.3) Preparation of intermediate sample plate: 4 μL of the 100× solution was added into a new 96-well plate, then 96 μL of the 1× kinase buffer was added, and the plate was shaken for 10 min for mixing evenly to serve as an intermediate sample plate.4) Preparation of test plate: 2.5 nL of the compound solution was taken from each well of the intermediate sample plate to a 384-well plate.(3) Kinase reaction1) The mTOR kinase was dissolved in the 1× kinase buffer to yield a 4× enzyme solution at a concentration that is 4 times the final concentration. 2.5 μL of the 2× enzyme solution was taken to each well of the test plate. The blank control without enzyme was added with 2.5 μL of the 1× kinase buffer instead of the enzyme solution. The test plate was shaken for mixing evenly.2) ULight-4E-BP1 polypeptide substrate and ATP were dissolved in the 1× kinase buffer to yield a 2× substrate solution at a concentration that is twice the final concentration. 5 L of the 2× substrate solution was taken to each well of the test plate. The test plate was shaken for mixing evenly.3) Kinase reaction:Each well of the test plate was covered and incubated at room temperature for 30 min.(4) Kinase detection1) The kinase quench buffer (EDTA) and Eu-anti-phospho-4E-BP1 antibody were formulated into a detection buffer at a concentration that is twice the final concentration. 10 μL of the detection buffer was added to each well of the test plate.2) The plate was centrifuged for a short time to mix evenly, shaken gently, and equilibrated at room temperature for 60 min.(5) Reading of the reaction wells(6) Calculation of inhibitory rates by means of curve fitting to the read values. |
| Affinity data for this assay | |
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