Equilibrium Dialysis vs. Ultracentrifugation for In Vitro Protein Binding Assays

In a modern drug discovery setting, in vitro protein binding assays are essential to assess the binding affinity and characteristics of a drug molecule with plasma proteins.

Two commonly used methods for in vitro protein binding assays are equilibrium dialysis and ultracentrifugation. Let’s compare these methods.

The Principle

Equilibrium Dialysis

The separation of free drug molecules from protein-bound drug molecules across a semipermeable membrane


The separation of free and protein-bound drug molecules based on the differences in sedimentation rate

The Process

Equilibrium Dialysis

Drug-protein mixture is placed in a dialysis chamber, and the chamber is immersed in a buffer solution

Over time, free drug molecules diffuse across the membrane into the buffer, while protein-bound drug molecules remain in the chamber


Drug-protein mixture is subjected to high centrifugal forces, leading to the formation of a sedimented pellet containing protein-bound drug molecules and a supernatant containing free drug molecules


Equilibrium Dialysis


Requires smaller sample volumes

Allows for the determination of binding constants and the characterization of drug-protein interactions


Minimize non-specific binding of the test compounds, provides accurate measurement of free drug fraction

Allows for the determination of binding constants and the measurement of drug-protein complex size


Equilibrium Dialysis

May suffer from potential experimental artifacts due to non-specific binding or low solubility at buffer side large sample volumes


Relatively low-throughput

Overall, the choice of method depends on the specific requirements of the drug discovery study.

Equilibrium dialysis is often preferred at early stage for screening purpose and has a relatively quick turnaround time. Ultracentrifugation provides better accuracy when the precise determination of binding constants and detailed characterization of drug-protein interactions are necessary.

It’s important to consider the advantages and limitations of each method when selecting the most suitable approach for in vitro protein binding assays for drug discovery, but our experienced scientists are here to help you make the right choice for your project.

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