Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | ADP Generation Assay |
| Description: | The primary assay for compound inhibitory activity was the ADP generation assay described herein. Test compounds were diluted to desired concentrations in kinase reaction buffer and briefly incubated with recombinant full-length human BTK kinase with a (His)6 tag (81.3 kDa; Invitrogen Corporation, Carlsbad, Calif.). The assay as described is based on volumes used in a standard 384 well format using solid, white-wall plates. The reaction was subsequently initiated by the addition of ATP and myelin basic protein (MBP) substrate (Millipore Corporation, Waltham, Mass.). Composition of the assay reaction mixture (5 mL volume) was: 5% v/v DMSO, 60 nM BTK, 1.6 uM ATP, and 20 uM MBP substrate. After incubation at room temperature for 60 min, 5 mL of the ADP-Glo reagent (Promega Corporation, Madison, Wis.) was added to each well and incubated for an additional 40 minutes. The reagent stopped the kinase reaction and depleted the unconsumed ATP. Kinase Detection reagent (10 mL; Promega Corporation) was then added to each well. The Kinase Detection reagent comprises reagents to convert ADP to ATP and provide luciferase and luciferin to detect ATP. Luminescence was measured on an EnVision microplate reader (PerkinElmer). The amount of luminescence from each reaction is directly correlated with BTK kinase activity. Percent inhibition and IC50 values were calculated by comparing enzyme activity in drug-treated wells to the appropriate controls. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |
|
Home |
| |
Search |
| |
Deposit |
| |
SiteMap |
| |
About us |
| |
Email us |
| |
Info |
|
©2000 BindingDB. All rights reserved. |
|||||||||||||