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| | Measurement of BTK Inhibitory Activity (In Vitro) |
| Description: | In a condition set for a method of measuring inhibitory activity in vitro of the compounds against BTK kinase activity, FL-Peptide 2 was used as a substrate, since it was described in a price list of LabChip (Registered trademark) series sample consumables of PerkinElmer Co., Ltd. that FL-Peptide 2 corresponded to a substrate peptide in a measurement of BTK kinase activity. The refined recombinant human BTK protein used in the test was purchased from Carna Biosciences, Inc.With regard to the measurement of the inhibitory activity of the compounds, firstly, monofumarate of Compound A was diluted stepwise with dimethyl sulfoxide (DMSO). Subsequently, BTK protein, a substrate peptide (final concentration was 1 μM), magnesium chloride (final concentration was 10 mM), ATP (final concentration was 45 μM), and a DMSO solution of the test compounds (final concentration of DMSO was 5%) were added to a buffer solution for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiotheitol, 0.01% Triton X-100), and after the solution was incubated for 40 minutes at 25° C., a kinase reaction was carried out. The reaction was terminated by adding EDTA thereto so as to obtain a final concentration of 30 mM. Finally, a substrate peptide that was not phosphorylated (S) and a phosphorylated peptide (P) were separated and detected by microchannel capillary electrophoresis with a LabChip EZ Reader II (PerkinElmer, Inc.). The amounts of phosphorylation reaction were determined from the individual peak heights of S and P, and the compound concentration at which the phosphorylation reaction could be suppressed by 50% was defined as the IC50 value (nM). |
| Affinity data for this assay | |
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