| Assay Method Information | |
| | Human Recombinant Btk Dissociation Dialysis Assay |
| Description: | A test compound was incubated with human recombinant Btk (100 nM) for 1.5 h at a concentration of 25 times the IC50 of Btk inhibition or 200 nM (whichever was greater). The incubation was performed in assay buffer (20 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM dithiothreitol, 50 μg/mL bovine serum albumen and 0.015% Brij 35). The reaction mixture was then dialyzed twice for 6 h each time against 1 L of assay buffer. The dialyzed reaction mixture (0.5 μL) was then diluted into a solution (100 μL) of ATP (2 mM) and substrate peptide (5 μM Src-tide, AnaSpec) such that the final Btk concentration was 1 nM (along with any inhibitor still bound). The assay was performed in matrix polypropylene 384-well plates. The reaction progress curve was monitored on the Caliper LABCHIP by electrophoretic separation of the substrate and phosphorylated product (pressure −1.2 psi, downstream voltage −500 V, upstream voltage −2300 V). Reaction velocity was measured over the linear phase and percent recovery of Btk activity was assessed at 2 h by comparing the fraction of phosphorylated peptide product relative to a DMSO-treated Btk control reaction containing no Example inhibitor. A control reaction with no Btk was also used to measure the background signal. A reversible inhibitor would show nearly complete recovery of Btk activity, while an irreversible inhibitor, would show little or no recovery of Btk activity. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |