| Assay Method Information | |
| | Btk Kinase Activity Assay |
| Description: | Specifically the following was added: (1) compound buffer or control: 5 μL of 5× compound buffer [(5× compound buffer comprised of: 1× Master Buffer, X μM test compound in 5% dimethyl sulfoxide; (2× Master Buffer comprised of 200 mM HEPES, pH 7.5, 0.2% BSA, and 0.02% Triton X-100)]; and (2) enzyme buffer: 10 L of 2.5× enzyme buffer (1× Master Buffer, 12.5 mM MgCl2, 2.5 mM DTT, 25 μM sodium orthovanadate, 25 μM beta-glycerophosphate, and 1.25 nM BTK enzyme). Human BTK enzyme Nanosyn-293HEK, wild-type, available from Nanosyn, Santa Clara, CA). Enzyme and compound were pre-incubated for 15 minutes. Additionally, the following was added: (3) substrate buffer: 10 μL of 2.5× substrate buffer (1× Master Buffer, 50 μM ATP, and 2.5 μM of the peptide substrate FAM). Each plate was incubated at 25° C. for 3 hours. The reaction was terminated by adding to each well: 45 L of 1.55× stop buffer (1× Master Buffer and 31 mM EDTA). The final reaction mixture was as follows: 100 mM HEPES, pH 7.5; 0.1% BSA; 0.01% Triton X-100; 1 mM DTT; 5 mM MgCl2; 10 μM sodium orthovanadate; 10 μM beta-glycerophosphate; 50 μM ATP; 1% dimethyl sulfoxide (from compound); 1 μM fluorescent peptide substrate and 0.5 nM BTK-enzyme. The terminated reactions were analyzed using a 12 channel LABCHIP® 3000 microfluidic detection instrument (available from Caliper Life Sciences, Waltham, MA). The enzymatic phosphorylation of the peptide resulted in a change in net charge, which enabled electrophoretic separation of product from substrate peptide. |
| Affinity data for this assay | |
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