BSEP Inhibition – Vesicles
This BSEP transporter assay is used to screen for inhibition of BSEP by a test compound in hBSEP-expressing vesicles at a single concentration
Required from Customer
- Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
- Exact molecular mass of test compound and its salt form
- Relevant solubility of the test compound
- MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.
- Normalized uptake of 3H-taurocholic acid in the presence and absence of test compound in hBSEP-expressing vesicles
- Percent inhibition by the test compound
- >50% inhibition indicates that the test compound is a significant inhibitor of BSEP
- BSEP probe substrate 3H-taurocholic acid at 1 μM
- Test compound at 10 μM in Hepes Influx Buffer
- hBSEP-expressing vesicles in the presence of ATP vs. AMP
- Measure uptake of 3H-taurocholic acid in hBSEP-expressing vesicles with and without test compound, in the presence of ATP vs. AMP
- Single concentration of test compound
- Four treatments as follows:
- hBSEP-expressing vesicles cells with 3H-taurocholic acid +/-test compound, in the presence of ATP and AMP (separately)
- Perform assay in duplicate (N=2 per treatment)
- Incubate cells for 30 min at 37°C
- Terminate the incubation
- Measure radioactivity of processed samples to determine 3H-taurocholic acid concentration
- Uptake rate of probe substrate in hBSEP-expressing vesicles in the presence of ATP vs. AMP
- The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
- The solubility of the test compound in Hepes Influx Buffer must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
- Percent inhibition is calculated as 100 x [1-(IRATP-IRAMP)TC / (IRATP – IRAMP)0], where IRATP is the influx rate of 3H-taurocholic acid in hBSEP-expressing vesicles in the presence of ATP, IRAMP is the influx rate of 3H-taurocholic acid in hBSEP-expressing vesicles in the presence of AMP, “TC” means in the presence of test compound, and “0” means in the absence of test compound.
- The customer can request:
- that a positive control be performed in parallel (additional fees apply)