Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | Inhibition Assay |
| Description: | PI3K enzymatic activity was assayed by measuring the amount of product phosphatidylinositol 3,4,5-phosphate (PIP3) formed from substrate 4,5 phosphatidylinositol 4,5-phosphate (PIP2) using a fluorescence polarization displacement assay. The decrease in fluorescence polarization of a fluorescent PIP.sub.3 probe is measured as it is displaced from a PIP.sub.3-binding protein GRP-1 detector by PI3K-catalyzed product. Assays were conducted in 384-well black Proxiplates in the presence of 10 mM Tris (pH 7.5), 50 mM NaCl, 4 mM MgCl.sub.2, 5% glycerol, 25 .mu.M ATP, 10 .mu.M PIP.sub.2 (Echelon Biosciences), 0.05% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM dithiothreitol, and 2% DMSO. The kinase reactions were initiated by the addition of 40 ng/mL p110.alpha./p85.alpha., 300 ng/mL p110.beta./p85.alpha., 40 ng/mL p110.gamma., or 40 ng/mL p110.delta./p85.alpha. (Upstate Group, Millipore; Dundee, UK), and 10 .mu.M PIP.sub.2 (Echelon Biosciences) to the wells. |
| 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. |
|||||||||||||