Assay Method Information

Assay Name:  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

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