Assay Method Information |
|
| Pharmacological In Vitro Assay |
Description: | Binding assays were performed in 96-well plate format, using a classical filtration assay with a human full length PPARγ construct (GST-PPAR LBD (25 μg/mL)) expressed in bacteria with some modifications regarding the conditionsof the experiments. The membrane-associated PPARγ was used as the biological source as previously described. Binding buffer consisted of 10 mM Tris/HCl, pH 8.2, containing 50 mM KCl and 1 mM dithiothreitol. Membrane preparations (5 μg/mL) were incubated for 180 min at 4°C in the presence of [3H]rosiglitazone (BRL49653, Amersham) (4 nM) and the tested compounds. Nonspecific binding was defined using an excess of unlabeled rosiglitazone (10 μM). Incubation was terminated by the addition of ice-cold 50 mM Tris/HCl buffer pH 7.4, followed by rapid filtrationunder reduced pressure through Whatman GF/C filter plates presoaked with ice-cold buffer, followed by three successive washes with the same buffer. Radioactivity was measured in a TopCount apparatus (Packard). |
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. |