| Assay Method Information | |
| | ADP-Glo Kinase Biochemical Assay |
| Description: | The activity of the Examples and Analogs described herein, as inhibitors of BTK are demonstrated and confirmed by pharmacological in vitro assays. Activity possessed by the compounds may be demonstrated in vivo. Those skilled in the art will appreciate that a variety of assay formats may be used to determine the activity of the compounds described herein.Materials:ADP-Glo Kinase Assay (cat. V9102, 10000 tests), components:1 50 ml ADP-Glo Reagent,1 100 ml Kinase Detection Buffer,1 Kinase Detection Substrate (Lyophilized),1 5 ml Ultra Pure ATP, 10 mM1 5 ml ADP, 10 mMReagents and Plate:Tris Hcl (Sigma cat. 154563), MgCl2 (Sigma cat. M1028), MnCl2 (Sigma, M3634), BSA (Sigma cat. 05470), BTK Substrate (Signalchem, P61-58), DTT (Sigma, D0632), DMSO (Sigma, S5879), BTK enzyme. (1.5 mg/ml, purity 75%, 90 ng/ul, made in house). 384 well assay plate (cat. 3674).Assay Conditions:Enzyme concentration: 8 ng/5 ul.ATP concentration: 50 uMSubstrate (peptide) concentration: 0.2 mg/ml.Reaction buffer composition: 40 mM Tris-HCl pH7.5, 10 mM MgCl2, 2 mM MnCl2, 0.1 mg/mL BSA, 0.05 mM DTT.Test compound concentration: DMSO≤0.5%.Methods:Compound Dosage Gradient Solution Preparation:A 3-fold serial dilution of test compound was made for 10 gradient points (100, 33.33, 11.11, 3.70, 1.23, 0.41, 0.14, 0.046, 0.015, 0.005 uM) in 100% DMSO. The intermediate dilution was done by adding 2 ul of diluted compound into 78 ul of assay buffer (containing 40 mM Tris Hcl, pH 7.5 10 mM MgCl2, 2 mM MnCl, 0.1 mg/mL BSA, 0.05 mM DTT), making the final compound concentration (1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15, 0.05 nM) and DMSO concentration 0.5% percentage. |
| Affinity data for this assay | |
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