| Assay Method Information | |
| | Human NK3 Receptor Binding Assay |
| Description: | CHO cells expressing hNK3 receptor were cultured in a MEMalpha medium (Nikken Seibutsu Igaku Kenkyusho, K.K.) containing 100 U/mL penicillin, 100 ug/mL streptomycin and 10% inactivated dialyzed serum. The medium was removed, the adhered cells were washed with PBS, and PBS containing 5 mM EDTA was added to detach the cells from the flask. The cells were collected by centrifugation, suspended in suspension buffer A (50 mM Tris-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 ug/mL chymostatin, 40 ug/mL bacitracin, 40 ug/mL APMSF, 1 mM EDTA), disrupted by a Polytron homogenizer (Kinematika), and centrifuged at 800xg for 10 min. The supernatant was recovered and ultracentrifuged at 100,000xg for 25 min. The precipitation fraction was suspended in suspension buffer B (50 mM Tris-HCl (pH 7.4), 0.02% bovine serum albumin, 2 ug/mL chymostatin, 40 ug/mL bacitracin, 40 ug/mL APMSF, 3 mM MnCl2), and cryopreserved (-80° C.) as a receptor reference standard. Measurement buffer (50 mM Tris-HCl (pH 7.4), 0.02% bovine serum albumin, 2 ug/mL chymostatin, 40 ug/mL bacitracin, 40 ug/mL APMSF, 3 mM MnCl2) (50 uL) was added to a 96-well microassay plate. The membrane reference standard (300 ug/mL) suspended in a measurement buffer was added by 50 uL. A measurement buffer containing 2% dimethyl sulfoxide was added by 50 uL to examine the total binding level, 16 uM non-labeled NK-B (PEPTIDE INSTITUTE, INC.) solution diluted with a measurement buffer containing 2% dimethyl sulfoxide was added by 50 uL to examine the non-specific binding level, and a test compound diluted with a measurement buffer (containing 2% dimethyl sulfoxide) was added by 50 uL to examine the binding inhibitory activity of the test compound. Furthermore, 400 uM [125I]-NK-B (Neurokinin-B (N-Me-Phe7), [125I]His3-) (PerkinElmer) solution was added to each well by 50 uL. After reaction at 25° C. for 30 min, the reaction was quenched using a cell harvester (PerkinElmer) by rapid filtration on a GF/C filter plate, and the cells were washed 5 times with 250 uL of a 50 mM Tris-HCl buffer (pH 7.4) containing 0.02% bovine serum albumin. The GF/C filter plate was dried, MicroScinti-0 (Perkin Elmer) was added by 20 uL, and the radioactivity was measured on a TopCount (PerkinElmer). The GF/C filter plate used had been immersed in 0.3% polyethyleneimine for one day. |
| Affinity data for this assay | |
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