Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | BTK FRET Competitive Binding Assay |
| Description: | The BTK time-resolved FRET-based competitive binding assay and cell-based BTK assays have been previously described.[Xu et al., J.Pharmacol. Exp. Ther., 341:90-103] For the BTK Omnia kinetic assay used in the mechanism of action experiments, assay reagents were prepared from the Omnia Y peptide 5 kit as follows: kinase reaction buffer was diluted to 1X in dH2O. A 10X substrate (100 μm) was prepared in dH20. ATP was prepared at 1 mM (10×) in dH2O, and 2 mM (10×) DTT was prepared in dH2O. A master mix of kinase buffer (10×), peptide (10×), and DTT (10×), all at 2 μL, and dH2O (4 μL) was prepared per well. Untagged BTK enzyme was run in a final concentration of 20 nM. Compounds at varying concentrations and ATP dilutions were prepared in 1V kinase buffer. Final compound DMSO concentration was 1.25 %. In a black non-binding plate (Corning 3676), enzyme (5 μL) was added to compound/ATP mix (5 μL). The plate was incubated for 20 min at 30 °C, then master mix (10 μL) was added into each well and mixed. The plate was incubated at 30 °C, and fluorescence intensity readings at λex=360 nm, λem=485 nm was collected at predetermined intervals on the Tecan M1000. |
| 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. |
|||||||||||||