| Assay Method Information | |
| | Scintillation Proximal Assay (SPA) |
| Description: | The scintillation proximal assay (SPA) was based on a competition binding assay with the radio-ligand N-(3-chloro-5-fluorophenyl-4-t)-4-nitrobenzo[c][1,2,5]oxadiazol-7-t-5-amine (1.8 TBq/mmol, affinity of the non-labeled ligand, IC50=82±18 nM, n=3). Assays were run using 384-well plates (781207/Greiner) in which one column was designated as the high signal control, and contained DMSO with no compound, and another column was designated as the low signal control, and contained no protein. Compounds (tested using a 14-point dose response with 3-fold compound dilutions from 100 μM to 60 pM) were pre-incubated for 30 min with HIF2α PAS-B domain (236-350, biotinylated on the N-terminus), before addition of the radio-ligand. Final concentrations in an assay volume of 60 μL were 5 nM HIF2α, and 25 nM radio-ligand. The assay buffer contained 50 mM Tris-HCl pH7 (Sigma), 20 mM NaCl (Fluka), 0.02% BSA (Sigma), 0.005% Triton X-100 (Pierce) and 1 mM DTT (Fluka). After a 30 min incubation period, 5 μL Streptavidin PVT SPA Beads (Perkin Elmer) at 1.2 mg/mL, diluted in the buffer, were added. After a 60 min incubation, plates were centrifuged and read on a Topcount NXT 384 (Packard). Duplicates were made using 2 different plates, and mean IC50 values were determined using the Helios system based on the following equation: % inhibition=[(high control−sample)/(high control−low control)]×100. The IC50 values in the Tabulated HIF2α activities are the average from 1 to 10 independent experiments. |
| Affinity data for this assay | |
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