Plasma Protein Binding

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Plasma Protein Binding Assay Information

Plasma protein binding (PPB) is an important parameter to measure as only the free unbound form of the drug is available to exert its pharmacological effect1.

The Rapid Equilibrium Dialysis (RED) system from Thermo Scientific™ is used to determine the protein binding of drugs in a high throughput assay using a stock solution of compound and plasma. The compound stock is added to plasma and is incubated in one side of a disposable insert and dialysis buffer added to the other side. Following an incubation period, samples are taken from both sides, quenched and analysed by LC-MS against a matrix-matched calibration curve.

Standard Assay Conditions

  • 100% plasma
  • Buffer: pH 7.4
  • 3 µM compound concentration
  • 4 hour incubation (N=2 replicates) at 37°C
  • Equilibrium check (N=1 replicate)
  • Matrix-matched calibration curve
  • Analysis by LC-MS
  • Marker compounds included
  • Various species/strains available


Excel summary of fraction unbound, percent bound, percent recovery, and equilibrium check.

Compound Requirements

100 µL of a 10 mM DMSO stock or 1 mg solid (accurately weighed to 2 decimal places).

Plasma Protein Binding

PPB in Rat and Human for 8 compounds, N=3 experiments each with N=3 replicates. Error bars denote standard deviation. 


  1. Smith et al (2010) The effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery, Nature Reviews, 9, 929-939.

Download Technical Resource Here

In Vitro Assays

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