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Rapid Equilibrium Dialysis Devices FAQ


Answers to frequently asked questions (FAQs) about Rapid Equilibrium Dialysis (RED) Devices


 

What is the membrane material in the RED Device Inserts?
Regenerated cellulose with a low glycerol content as a humectant.

What is the pore size or molecular weight cut-off (MWCO) for the membrane?

The nominal MWCO is 6,000-8,000 Daltons as specified by the manufacturer.

Is there any preprocessing, such as rinsing the RED Device Inserts, which is required before they can be used?
No. The inserts can be used directly out of the package, no presoaking or rinsing of the membrane is needed.

Are the units sterile or endotoxin free?
The units do not undergo a sterilization procedure nor are they tested for endotoxin content.

How long does it take to reach equilibrium dialysis?
In most cases 4 hours is sufficient to reach equilibrium. This is due to the high surface area to volume ratio for the membrane (7.4:1).

Why is the volume different between the membrane chamber and the buffer chamber? Will it affect the results?
The difference in liquid volume is to maintain the same level (height) between the two chambers. The equilibrium constant depends only on the concentration of the ligand, not the volume.

Is there a volume shift in either sample?
Volume shift in the plasma is unusual due to how quickly equilibrium can be reached with the RED Device. If much longer incubation times are used, there can be an increase in the volume of the plasma. However, the ligand will still reach equilibrium, having free ligand at the same concentration on both sides of the membrane.

Is there any non-specific binding of compounds to the device?
The container for the device is PTFE and the insert is made of high density polyethylene (HDPE) and are highly hydrophobic. The regenerated cellulose membrane is a standard material for commercial dialysis devices. A recovery study shows a consistency of 85% recovery of high and low protein binding compounds. This result is indicative of minimal non-specific binding.

What samples are typically tested for binding of ligands with the RED Device?
Plasma samples from human, mouse and rat. For toxicology studies, monkey is also typically tested. Typically pooled plasma samples purchased from commercial vendors are used, although researchers could test the differences in plasma from various physiological states using the RED Devices.

What is the minimum volume of plasma that I can use?
The lowest validated volume for the assay is 200µl of plasma. We are in the process of evaluating 100µl plasma samples.

Can the RED Devices be reused?
No, these are designed for a single use, as plasma is sticky and cannot be removed from the unit. The PTFE plate is designed for multiple uses.

If we use radioactive compounds in our study, will the PTFE plate be contaminated, preventing reuse?
No. After use, rinse the PTFE plate in 20% ethanol in water and rinse with ultrapure water at least two times. No radioactivity will remain after these rinses.

If my compound has low solubility in aqueous buffer, can I use DMSO and if so, at what concentration?
A final concentration of 1% DMSO is acceptable and will not affect the study. Have we tested DMF and other solvents? No, DMF has not been tested. However, it is expected that DMF should be similar to DMSO in terms of use.

How do I mix the samples to speed reaching equilibrium?
Once samples are loaded into the PTFE plate it should be placed on a shaking device. With an orbital shaker 100 rpm works well. For an up and down shaker, 20 rpm is sufficient.

If I see vapor condensing on the clear sealing film, will the loss of water vapor affect the result?
No. It will not affect the equilibrium.

Can I heat the PTFE block on a hot plate to maintain the temperature?
Yes, as most assays are performed at physiological temperature (37.5°C).

Why can I only process 48 samples on a 96-well plate?
Each sample requires two chambers for equilibrium dialysis to take place (96/2 = 48).

 

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