Tissue Binding assay
Aim: Screening of in vitro tissue binding of test compound in tissue homogenate from rodents, using Rapid Equilibrium Dialysis (RED).
Background information: Tissue binding determines the amount of free compound available at the action site. The extent of binding to tissue components influences the efficacy and/or toxicological effects of drugs.
Equilibrium dialysis is the most widely accepted method for assessing plasma and tissue binding as non-specific binding effects are minimized compared to other methods such as ultrafiltration.
The RED device consists of disposable inserts and a base plate. Each insert is made of two side-by-side chambers separated by a vertical cylinder of a semi-permeable dialysis membrane with minimal nonspecific binding. The system is allowed to equilibrate at 37ºC. The amount of test compound present in each compartment is quantified by UPLC-MS/MS.
Compound requirement: 150 µl of a 10 mM DMSO solution
Turnaround time: 1 week / 2 compounds
- Test compound concentration: 5 µM (variable on request)
- Number of replicates: 3
- Dilution factor of tissue homogenate: 5 (in saline phosphate buffer)
- Equilibrium dialysis conditions: 37ºC for 4 h
- Dilution with blank tissue homogenate (buffer sample) or buffer (tissue homogenate sample)
- Stability in tissue homogenate checked in parallel
- Analytical method: UPLC-MS/MS quantification of tissue and buffer samples after protein precipitation