| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence Assay |
| Description: | The inhibitory properties of compounds to ASK1 may be determined using a white 384-well-plate format under the following reaction conditions: 25 nM ASK1, 1 uM CisBio STK S3-biotion peptide, 100 uM ATP, and 1%-2% DMSO in kinase assay buffer of 50 mM HEPES, pH 7.3, 10 mM NaCl, 10 mM MgCl2, 0.01% Brij35, 0.2 mM EDTA, and 1 mM DTT. Reaction product is determined quantitatively by HTRF after the addition of detection reagent SA-XL665 and STK-antibody-cryptate.The assay reaction may be initiated as follows: 2 ul of the mixture of 3 uM CisBio STK S3-biotion peptide and 300 uM ATP with 2 ul of test compound (2 fold serial dilutions for 11 data points for each inhibitor) containing 3%-6% DMSO are added to each well of the plate, followed by the addition of 2 uL of 75 nM ASK1 to initiate the reaction (final enzyme concentration was 25 nM for ASK1). The reaction mixture may then be incubated at room temperature for 1 hour, and quenched and developed. |
| Affinity data for this assay | |
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