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Assay Method Information |
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| | BCA Protein Assay |
| Description: | The buffer solution for the receptor binding test was dispensed to the wells of a 96-well assay plate (Greiner) at 22.5 uL/well. DMSO solutions of a test compound, which were prepared at an 80-time higher concentration using 100% dimethyl sulfoxide (DMSO), were added to the wells at 2.5 uL/well (final concentrations of 1 nM to 100 nM), and the solutions were mixed. As a radiolabeled ligand, 125I-substance P (Substance P, [125I]Tyr8-, PerkinElmer) was used. 125I-substance P was diluted with the buffer solution for the receptor binding test to a concentration resulting in 125 pmol/25 uL/well and added to the 96-well assay plate, and the solutions were mixed. The membrane fraction prepared from the human NK1 receptor-expressing cells was diluted with the buffer solution for the receptor binding test to a concentration resulting in 8 to 10 ug/well, suspended until the suspension became in such a homogenous state that the suspension could flow through as 27G injection needle smoothly and then added to the 96-well assay plate at 150 uL/well. Then, the plate was incubated at room temperature for 60 minutes while shaking the plate. The reaction solutions were suction-filtered. through a multiscreen 96-well filter plate (Millipore) which had been pre-treated with 0.3% polyethyleneimine, and the reaction was terminated by washing with a washing solution (50 mM Tris and 0.02% bovine serum albumin, pH 7.4) four times. The bottom of the microplate was dried at 60° C., and then 100 uL/well of MicroScint 20 (PerkinElmer) was dispensed to the wells. The top of the plate was sealed with TopSeal A (PerkinElmer), and the plate was shaken for 5 to 10 minutes. Then, the radioactivities were measured with TopCount NXT (registered trademark) (PerkinElmer). The radioactivity of each well was calculated by subtracting the radioactivity of the well to which 10 uM aprepitant was added (non-specific binding). The binding rate (%) of 125I-substance P=(the radioactivity of the group to which the test compound was added)/(the radioactivity of the group to which the vehicle was added)x100 was calculated. Using analysis software, GraphPad Prism (GraphPad Software), the binding rate (%) was plotted against the concentration of the test compound and linearly approximated, and the concentration required for 50% inhibition, IC50, was calculated. |
| Affinity data for this assay | |
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