EA277

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.

Deliverables

  • 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

Substrate

  • BSEP probe substrate 3H-taurocholic acid at 1 μM
  • Test compound at 10 μM in Hepes Influx Buffer

Assay System

  • hBSEP-expressing vesicles in the presence of ATP vs. AMP

Assay Conditions

  • 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

Assay QC

  • Uptake rate of probe substrate in hBSEP-expressing vesicles in the presence of ATP vs. AMP

Notes

  1. 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.
  2. 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.
  3. 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.

Options

  1. The customer can request:
    • that a positive control be performed in parallel (additional fees apply)

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