| Assay Method Information | |
| | Radioligand Binding Assay |
| Description: | i. Prepared the assay buffer following the table below; Reagent Concentration Tris 50 mMCaCl2, 4 mMBSA, 0.1% (w/V) Adjust pH to 7.4 followed by 0.2 μM sterile filtration ii. Preparation of 8 doses of reference and test compounds starting from 10 mM stock solution as requested by 5-fold serial dilutions with 100%; iii. Prepared (v/v) DMSO: a. 50 μl/well of 0.5% (v/v) PEI was added to UniFilter-96 GF/B plates. The plates were sealed and incubates at 4° C. for 3 hrs; b. After incubation, the plates were washed 3 times with ice-cold wash buffer (50 mM Tris, pH7.4); iv. Preparation of assay plates: a. Cell membrane were diluted with assay buffer and 330 μl/well was added to 96 round deep well plates to reach a concentration of 20 μg/well; b. 8 concentrations of reference or test compounds were prepared and 110 μl/well was added to 96 round deep well plates; c. [3H]-ketanserin was diluted with assay buffer to 5 nM (5× final concentration) and 110 μl/well was added to 96 round deep well plates. v. The plate was centrifuged at 1000 rpm for 30 secs and then agitated at 600 rpm, R.T. for 5 min. vi. The plates were sealed and incubates at 27° C. for 90 min.vii. The incubation was stopped by vacuum filtration onto GF/B filter plates followed by 4 times washing with ice-cold wash buffer (50 mM Tris, pH7.4).viii. The plates were dried at 37° C. for 45 min. ix. The filter plates were sealed and 40 μl/well of scintillation cocktail was added. x. The plate was read by using a Microbeta2 microplate counter. |
| Affinity data for this assay | |
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