| Assay Method Information | |
| | Electrophoretic Mobility Shift Assay (MSA) |
| Description: | Btk kinase activity was measured in vitro using an electrophoretic mobility shift assay (MSA). The ability of Btk to phosphorylate a fluorescent peptide substrate (FAM-GEEPLYWSFPAKKK-NH2) was measured. The kinase reactions were assembled in a total volume of 25 μL per well in 384 well plates. The following was added to each well: compound buffer (or control); enzyme buffer; and substrate buffer, as further described below. Specifically the following was added: (1) compound buffer or control: 5μL 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, pH7.5, 0.2% BSA, and 0.02% Triton X-100)]; and (2) enzyme buffer: 10 μL 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 μL of 1.55× stop buffer (1× Master Buffer and 31 mM EDTA). The final reaction mixture was as follows: 100 mM HEPES, pH7.5; 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); 1μM fluorescent peptide substrate and 0.5 nM BTK- enzyme. |
| Affinity data for this assay | |
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