| Assay Method Information | |
| | Affinity for histamine H4-receptor was determined by H4 Binding assay |
| Description: | Membranes from CHO-K1 cells transfected with human H4 receptor and microplates of 96-well format were used.For competition studies cell membranes were homogenated in Tris-HCl 50 mM, EDTA 5 mM, pH 7.4 and added to the microplate at the concentration of approximately 15 μg/well. Membrane suspensions were incubated with test compounds for 15 min at 25° C. and binding reaction was initiated by the addition of the specific H4 radioligand [PH] histamine at a final concentration of 5-8 nM (KD=11.1 nM; Bmax=4,13 pmol/mg protein). Non specific binding was defined using 10 μM unlabeled histamine and the total incubation volume was 275 μl per well.Incubations were performed under gentle agitation at 25° C. for 60 min and concluding with a rapid vacuum filtration using pre-soaked filters (0.5% polyethylenimine). After ten rapid washes with cold wash buffer (50 mM Tris-HCl buffer) the filter plates were dried at room temperature for 30 min.Finally, scintillation liquid (Microscint 20) was added to the whole microplate in order to measure the radioactivity reattained on the filters using a specific scintillation counter (Top Count—NXT). Curve analysis and values of IC50 were determined using Grap Pad Prism program (GraphPad Software, San Diego, USA).Test compounds were initially evaluated at one concentration (1 μM) and thereafter if significant displacement was observed (>40%) a concentration-response curve was conducted. Each test concentration was measured in triplicate. |
| Affinity data for this assay | |
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