| Assay Method Information | |
| | In Vitro Assay |
| Description: | I. Prepare 1× Kinase Base Buffer and Stop Buffer for Testing Kinases1) 1× Kinase base buffer (for TRK-A)50 mM HEPES, pH 7.50.0015% Brij-352) Stop buffer100 mM HEPES, pH 7.50.015% Brij-350.2% Coating Reagent #350 mM EDTAII. Prepare Source Plate with Compound1) Dilute the compound to 50× of the final concentration in reaction by 100% DMSO. Transfer 100 μl of this compound dilution to a well in a 96-well plate. For example, if the desired highest inhibitor concentration is 10 μM, then prepare a 500 μM of compound DMSO solution in this step.2) Perform 3× series dilution of the compound dilution obtained in step 1) with 100% DMSO to provide 9 more diluted solutions of the compound.3) Add 100 μl of 100% DMSO to two empty wells for no compound control and no enzyme control in the same 96-well plate. Mark the plate as source plate.4) Prepare intermediate plateTransfer 10 μl of compound from the source plate to a new 96-well plate as the intermediate plate.Add 90 μl of 1× Kinase base buffer to each well of the intermediate plate.Mix the compounds in intermediate plate for 10 mM on a shaker.5) Prepare assay plateTransfer 5 μl of each well from the 96-well intermediate plate to a 384-well plate in duplicates. For example, A1 of the 96-well plate is transferred to A1 and A2 of the 384-well plate. A2 of the 96-well plate is transferred to A3 and A4 of the 384-well plate, and so on.III. Kinase Reaction1) Prepare 2.5× enzyme solution Add kinase in 1× kinase base buffer.2) Prepare 2.5× peptide solutionAdd FAM-labeled peptide (SEQ ID NO:1) and ATP in the 1× kinase base buffer.3) Transfer 2.5× enzyme solution to the assay plateAssay plate already contains 5 μl of the compound in 100% DMSO.Add 10 μl of 2.5× enzyme solution to each well of the 384-well assay plate.Incubate at room temperature for 10 min4) Transfer 2.5× peptide solution to the assay plateAdd 10 μl of 2.5× peptide solution to each well of the 384-well assay plate.5) Kinase reaction and stopIncubate at 28° C. for a specified period of time.Add 25 μl of Stop buffer to stop reaction.IV. Caliper ReadingCollect data on Caliper.V. Curve Fitting |
| Affinity data for this assay | |
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