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| | In vitro [3H]histamine Binding Assay |
| Description: | Prior to the experiments, cell membranes were sedimented by a 10 min centrifugation at 4 °C and 16 0009x g and resuspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA and 75 mM Tris/HCl, pH 7.4). Competition binding experimentswere carried out by incubating membranes, 35 ug/well (prepared from Sf9 cells transiently expressing human H4R, co-expressed with G proteins Galphai2 and Gbeta1gamma2 subunits) in a final volume of 0.2 mL containing binding buffer and [3H]histamine 2x HCl (10 nM, 15.3 Ci/mmol, PerkinElmer, Waltham, MA, USA) in a 96 well microtitre plate. Assays were run in duplicates or triplicates with four or seven appropriate concentrations between 100 nM or 0.1 nM and 100 uM of the test compounds. Amount of experiments was at least 2. Only exceptions were for substance 2, 3, and 4 where only one experiment in duplicates was performed.Incubations were performed for 60 min at 25 °C and shaking at 250 rpm. Non-specific binding was determined. |
| Affinity data for this assay | |
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