Equilibrium dialysis is a frequently used technique for measuring compound-plasma protein binding. The Thermo Scientific Rapid Equilibrium Dialysis (RED) Device is an excellent device for conducting this analysis because it produces accurate and consistent data with shorter preparation and dialysis times.
Because only the unbound (free) fraction of a compound elicits the desired response/reaction, its binding to plasma proteins has a significant impact on compound pharmacokinetics and pharmacodynamics. Therefore, the unbound fraction of the compound is a crucial parameter that is used, alongside other laboratory analyses, for optimizing compound efficacy.
The RED Device uses a size-defined dialysis membrane to quantitate active drug molecules that are not bound to plasma proteins in an equilibrium state. This device is validated for plasma binding assays, producing results consistent with those reported in the literature.
How does the RED Device work?
The RED device consists of a disposable polypropylene base plate and disposable dialysis inserts. Each insert is divided into 2 side-by-side compartments separated by a dialysis membrane (8K or 12K MWCO). For these ready-to-use devices, simply add the sample and buffer to the appropriate chambers, then incubate to enable the separation of free, non-specific protein (Figure 1).
Figure 1. The RED Device mechanism of action.The RED Device uses equilibrium dialysis for assessing the amount of compound-protein binding. The insert chambers of the device are separated by a semi-permeable membrane and, when filled with sample, enable the separation of the free compound fraction from that of compound bound to plasma protein.
Which RED device is best for your experiment?
| Single-Use Red Device, 8K | Red Device Inserts, 8K | Single-Use Red Device, 12K | Competition RED Inserts | |
|---|---|---|---|---|
| Application | Plasma protein binding studies | Drug partioning between plasma and tissue | ||
| MWCO | 8K | 8K | 12K | 8K |
| Pre-loaded inserts | ✓ |
| ✓ | |
| Disposable plate included | ✓ |
| ✓ | |
| Recommended for variable # of samples per run | ✓ | ✓ | ||
| See all Rapid Equilibrium Dialysis (RED) Devices | ||||
Recommended accessories:
Pierce Protein Precipitation Plates—96-well format for protein precipitation; ideal for downstream processing of plasma protein binding assay samples prepared with RED systems
Video of how the RED Device works
How to use the Rapid Equilibrium Dialysis (RED) Device
The RED Device is easy to use. Simply load the spiked sample and buffer into their respective chambers. Incubate the contents on a shaker for up to 4 hours, and then transfer them for the end-point assay. View the instructional video showing how to use the RED device.
RED device featured data
| Device | Time to reach Equilibrium | Leakage | Disposable | Labor Intensity | Automation Accessible | Vol. Shift |
|---|---|---|---|---|---|---|
| Multi-Equilibrium DIALYZER (Dianorm, Harvard, Spectrum) | 3–4 h | Some | No | ++++ | No | Minimum |
| 96-Well Equilibrium DIALYZER†† (Harvard Apparatus) | 16 h | Some | Yes | +++ | Possible | Yes |
| 96-Well Micro-Equilibrium Dialysis (HTDialysis, LLC) | 6 h | Some | No | +++ | Possible | Yes |
| RED Device (Thermo Fisher Scientific) | 4–6 h (2 h) | None | Yes | + | Yes | None |
Plasma protein-binding results for seven compounds using the Thermo Scientific RED Device 100-minute protocol are equivalent to results achieved using classic devices found in the literature. A time course study using vortex agitation at 750 rpm and 37°C was used to determine the free fraction at equilibration.
Comparison of in vitro and in vivo tissue/plasma binding ratios of Taxol in rat. Presence of low Taxol in brain tissue in vivo indicates the blood-brain barrier inhibits movement of Taxol to the brain.
| Device | Time to reach Equilibrium | Leakage | Disposable | Labor Intensity | Automation Accessible | Vol. Shift |
|---|---|---|---|---|---|---|
| Multi-Equilibrium DIALYZER (Dianorm, Harvard, Spectrum) | 3–4 h | Some | No | ++++ | No | Minimum |
| 96-Well Equilibrium DIALYZER†† (Harvard Apparatus) | 16 h | Some | Yes | +++ | Possible | Yes |
| 96-Well Micro-Equilibrium Dialysis (HTDialysis, LLC) | 6 h | Some | No | +++ | Possible | Yes |
| RED Device (Thermo Fisher Scientific) | 4–6 h (2 h) | None | Yes | + | Yes | None |
Plasma protein-binding results for seven compounds using the Thermo Scientific RED Device 100-minute protocol are equivalent to results achieved using classic devices found in the literature. A time course study using vortex agitation at 750 rpm and 37°C was used to determine the free fraction at equilibration.
Comparison of in vitro and in vivo tissue/plasma binding ratios of Taxol in rat. Presence of low Taxol in brain tissue in vivo indicates the blood-brain barrier inhibits movement of Taxol to the brain.
Why test for plasma protein binding?
Screening for adsorption, disposition, metabolism, and elimination (ADME) includes measuring for plasma protein binding when testing compounds. Knowing the amount of compound binding to the plasma protein provides guidance in pharmacokinetic distribution. Bound compound may impact efficacy due to the amount of free compound available for target-binding, as well as tissue distribution. The RED Device helps accelerate testing for plasma protein binding during the pre-clinical process by being semi-automated, ready-to-use, and enabling quick cleanup with radioactive material.
- Ye Z, Zetterberg C, Gao H. Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan workstation. J Pharm Biomed Anal. 2017 Jun 5;140:210-214. doi: 10.1016/j.jpba.2017.03.019. Epub 2017 Mar 14. PMID: 28365514.
- National Center for Biotechnology Information. "PubChem Bioassay Record for AID 1937, Source: Burnham Center for Chemical Genomics" PubChem, https://pubchem.ncbi.nlm.nih.gov/bioassay/1937. Accessed 22 March, 2023.
- Waters NJ, Jones R, Williams G, Sohal B. Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding. J Pharm Sci. 2008 Oct;97(10):4586-95. doi: 10.1002/jps.21317. PMID: 18300299.
- Moein MM, Halldin C. Sample preparation techniques for protein binding measurement in radiopharmaceutical approaches: A short review. Talanta. 2020 Nov 1;219:121220. PMID: 32887121.
- Shave, S.; Pham, N.T.; Śmieja, C.B.; Auer, M. Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties. Methods Protoc. 2020, 3, 55.
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