| Assay Method Information | |
| | In Vitro Radioligand Binding Assay |
| Description: | In Vitro Radioligand Binding Studies for Determination of the Dissociation Constants K1 at the Human Adrenoreceptor ADRA2C (Eurofins Panlabs Discovery Services, Taiwan, Ltd)A competition assay based on [3H] rauwolscine as radioliganden was used to determine the binding affinity of the test substances at the human ADRA2C receptor.To configure the competition assay, the equilibrium dissociation constant Kd of the radioligand [3H] rauwolscine was determined in a saturation experiment. To this end, homogenates of CHO-K1 cells recombinantly expressing the human ADRA2C receptor were incubated with increasing concentrations of the radiotracers for 1 h at 4° C. in binding buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.4). Unspecific binding was determined by addition of an excess of the not radioactively labelled ligand prazosin (10 μM). The radioactivity was determined in a scintiation counter.The competition experiments were carried out in the presence of 0.5 nM [3H] rauwolscine and increasing concentrations of the test substances to be characterized under the conditions described above. The substance concentration which displaces 50% of the radiolabelled ligand is referred to as IC50 value. |
| Affinity data for this assay | |
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