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Assay Method Information |
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| | Binding Assay |
| Description: | Each compound of the present invention prepared in the examples was prepared in DMSO into concentrations of 0.02, 0.06, 0.3 and 2 mM. 10 uL of the prepared compound solution was allocated in each well of a 96-well plate, and then 10 uL of 200 uM (R)( )-α-methylhistamine (RaMH) diluted with analysis buffer and 1% of DMSO were transferred to each well to calculate non-specific binding and total binding degree. 15 ug of human histamine 3 receptor-overexpressed cell membrane (PerkinElmer) was diluted with 180 uL of analysis buffer solution (50 mM tris-HCl pH 7.4, 5 mM MgCl2), and that was transferred to each well. [3H] labeled Na-methylhistamine (PerkinElmer) was diluted into 20 uM concentration, 10 uL of that was added to each well, and then it was kept in a 27 ° C. incubator for 30 minutes. After the reaction, 200 uL of the mixture was transferred to a glass fiber plate in which 0.5% polyethylene amine was presoaked, and then non-binding [3H] labeled Na-methylhistamine was removed in vacuum. After 5 times washing with 200 uL of washing buffer solution (50 mM tris-HCl, pH 7.4), the plate was dried in a 37 ° C. oven for 18 hours. 100 uL of betascint cocktail solution was added to each well, and after 1 hour CPM of [3H] labeled N-α-methylhistamine was measured by using Wallac beta-counter TriLux. |
| Affinity data for this assay | |
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