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| | Radioligand Binding Assay |
| Description: | Radioligand binding was performed using filtration or scintillation proximity assay (SPA) technology. SPA assays were done at room temperature in 10 mM HEPES pH 7.4, 1 mM EDTA containing 2 mM MnCl2 and 16 nM [3H]-PGD2 (PerkinElmer, Waltham, Mass.) (164 Ci mmol-1), in a final volume of 0.05 mL. Competing ligands were diluted in dimethylsulfoxide and added using very low volumes (500 nL). The reaction was initiated by the addition of a mixture of 3.52 ug of membrane protein prepared from a human embryonic kidney (HEK)-hCRTH2 cell line adhered to 140 ug of wheatgerm agglutinin SPA beads (PerkinElmer). Total and non-specific binding were determined in the absence and the presence of a CRTH2 antagonist, respectively. The reaction was routinely conducted for 60 minutes at room temperature followed by centrifugation for 5 minutes at 1000 RPM. The radioactivity was measured with a TopCountNXT (PerkinElmer). Filtration binding assays were done in a similar way with minor differences. The assay volume was 0.20 mL and competing ligands in dimethylsulfoxide were added in 2 uL. [3H]-PGD2 was used at 0.6 nM, and reactions were initiated by the addition of 10 ug of membrane protein. Reactions were terminated by filtration through GF/C filter plates (PerkinElmer) presoaked in 10 mM HEPES. After washing with buffer, plates were dried in a 50° C. oven for 1 h, scintillation cocktail was added and radioactivity was measured. Results determined using the SPA-based assay were similar to those from the filtration binding assay. |
| Affinity data for this assay | |
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