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| | Radioligand Binding Assay II |
| Description: | For crude cell membrane preparations, cells (CHO, Chinese hamster ovary or HEK, human embryonic kidney) expressing human orexin 1 or human orexin 2 receptors, were washed with HEPES (10 mM, pH 7.5), scraped off the culture plates with the same buffer, and centrifuged at 4 C for 5 min at 2500 x g. The cell pellet was either stored at -80 C or used directly. Before the experiments, cell membranes were re-suspended in binding assay buffer (10 mM HEPES, 0.5% (w/v) bovine serum albumin, pH 7.5) by homogenisation with a Polytron homogeniser at 50 Hz for 20 s. In competition experiments, cell homogenates (150μΙ) were incubated in assay buffer (10 mM HEPES, pH 7.5, 0.5 % (w/v) bovine serum albumin, 5 mM MgCI2, 1 mMCaCI2, and tween 0.05%) for 1 h at room temperature with about 1 nM of the radioligand [3H]-SB649868, 66 Ci/mmole, 50 μΙ), and with various concentrations of compounds of the invention (50 μΙ) in triplicates. |
| Affinity data for this assay | |
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