Reacción #463670

Ecuación de reacción

Tris

dithiothreitol

β-mercaptoethanol

leupeptin

benzamide

thiazolidinedione

EDTA

PMSF

→

[3H]AD-5075

Disolventes

glycerol

Condiciones de reacción

Tratamiento posterior

1. 1
   workup.ADDITIONcontaining 5-10% COS-1 cell cytoplasmic lysate and 10 nM
2. 2
   workup.WAITAssays were incubated for ~16 h at 4 ° C.
3. 3
   OtroUnbound ligand was removed by addition of 200 μl dextran/gelatin-coated charcoal, on ice, for ~10 minutes

Procedimiento

For each assay, an aliquot of receptor (1:1000-1:3000 dilution) was incubated in TEGM (10 mM Tris, pH 7.2, 1 mM EDTA, 10% glycerol, 7 μl/100 ml β-mercaptoethanol, 10 mM Na molybdate, 1 mM dithiothreitol, 5 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml benzamide and 0.5 mM PMSF) containing 5-10% COS-1 cell cytoplasmic lysate and 10 nM labeled thiazolidinedione ([3H2 ]AD-5075, 21 Ci/mmole), ± test compound compound, [3H2 ]Example 11, 17 Ci/mmole), ± test compound, respectively. Assays were incubated for ~16 h at 4 ° C. in a final volume of 300 μl. Unbound ligand was removed by addition of 200 μl dextran/gelatin-coated charcoal, on ice, for ~10 minutes. After centrifugation at 3000 rpm for 10 min at 4° C., 200 μl of the supernatant fraction was counted in a liquid scintillation counter. In this assay the KD for AD-5075 and Example 11 is 1 nM, respectively.

Fuente