This assay is used to screen for inhibition of MATE2K by a test compound in MATE2K-transfected HEK293 cells at a single concentration

Catalog number EA288

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 Metformin in the presence and absence of test compound in MATE2K-transfected and vector control cells
  • Percent inhibition by the test compound
  • >50% inhibition indicates that the test compound is a significant inhibitor of MATE2K

Substrate

  • MATE2K probe substrate Metformin at 50 μM
  • Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

  • MATE2K-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

  • Measure uptake of Metformin in MATE2K-transfected and vector control cells with and without test compound
  • Single concentration of test compound
  • Two cell lines: MATE2K-transfected and vector control cell lines
  • Four treatments as follows:
    • MATE2K-transfected cells with Metformin +/-test compound
    • Vector control cells with Metformin +/- test compound
  • Perform assay in duplicate (N=2 per treatment)
  • Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
  • Buffer is aspirated and replaced with test compound dosing solution
  • Incubate cells for 5 min at 37°C
  • Terminate the incubation and lyse the cells
  • Analyze cell lysate using LC-MS/MS to determine Metformin concentration
  • Determine protein concentration in cell lysate and normalize Metformin concentration to protein content

Assay QC

  • Uptake rate of probe substrate in MATE2K-transfected vs. vector control cells

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 Hanks’ buffer containing < 0.8% DMSO 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-(IR1B1-IRVC)TC / (IR1B1 – IRVC)0], where IR1B1 is the influx rate of Metformin in MATE2K-transfected cells, IRVC is the influx rate in vector control cells, “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)