Fraction Unbound (Rat or Mouse Brain)

This assay uses equilibrium dialysis to determine the unbound fraction of test compound in a rat or mouse brain homogenate, a parameter that helps predict brain penetration in vivo.

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
  • MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc.


  • The calculated percent unbound of test compound and control compound


  • Test compound at a single concentration (5 μM) in rat or mouse brain homogenate at pH 7.4 with DMSO concentration less than 1%

Assay System

  • 96-well equilibrium dialysis or equivalent apparatus at 37°C
  • Rat or mouse brain homogenate (≥ 3 male donors  pooled) in phosphate-buffered saline (PBS) at pH 7.4

Assay Conditions

  • Adjust pH of PBS to 7.4 prior to initiation of the experiment
  • Perform equilibrium dialysis in duplicate (N=2)
  • Homogenize brain tissue in 2 volumes of PBS
  • Spike test compound into diluted brain homogenate (final concentration 5μM)
  • Dialyze the test compound in brain homogenate against PBS at 37°C
  • Sample brain homogenate and buffer in each well at >22 hours after the system reaches 37°C
  • Normalize samples to achieve the same analytical matrix
  • Use LC-MS/MS to determine the peak area response ratio for the test compound and control compound (relative to the peak area of an analytical internal standard) without running a calibration curve

Assay QC

  • Co-dose test compound with a control compound with low fraction unbound

Dynamic Range

  • Up to 99.0% bound


  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. This assay uses a frozen/thawed rat brain homogenate.
  3. Results from this assay, when combined with results from EA702, ExpressPlus Plasma Protein Binding (Rat Plasma) and EA203, ExpressPlus BBB Penetration Potential, may be used to predict brain permeation in vivo.