| Assay Method Information | |
| | hB0AT1 Inhibitory Test |
| Description: | A test compound dissolved in dimethyl sulfoxide (DMSO) was added to a 96 well plate for solid phase radioactivity measurement containing a scintillator at the bottom of the well (final concentration of DMSO 0.5%). DMSO alone as a control and DMSO containing N-(4-bromophenyl)-3,5-dichloro-2-hydroxybenzamide (final concentration 10 M) for the is measurement of B0AT1 non-specific uptake were similarly added at 0.5 μL per well. Human B0AT1 stable expression CHO cells suspended in a buffer (buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 0.01% bovine serum albumin, and 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) were added by 90 μL to 75000 cells/well. After standing at ordinary temperature for 30 min or longer, the cells were subjected to a phenylalanine uptake experiment. A buffer containing L-phenylalanine and [3,4,5-3H]-L-phenylalanine was added by 10 μL per well (final concentration of phenylalanine 0.25 mM), and the radioactivity of the plate bottom surface at ordinary temperature was measured with a scintillation counter over time, and the radioactivity value after 100 to 200 min was analyzed. A value obtained by subtracting the B0AT1 non-specific uptake from the control uptake was taken as 100%, and the concentration necessary for each test compound to achieve 50% inhibition (IC50 value) was determined by nonlinear regression using a logistic model. |
| Affinity data for this assay | |
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