Dialysis - Knot the Way to Do It

There are certain things that you should not do when performing dialysis in the laboratory. To ensure that all unwanted small molecular weight substances (such as reducing agents, non-reacted crosslinking or labeling reagents, and preservatives) are removed and facilitate a more efficient buffer exchange for macromolecular samples such as proteins, here are some of the things you need to avoid at all cost.   

Preparation and Handling of Dialysis Membrane Tubing

Most of the membranes used for laboratory dialysis are made of regenerated cellulose. During the manufacturing process, glycerol is added to prevent cracking during the drying process and to help maintain the desired pore structure. To remove glycerol and other low level contaminants such as sulfur compounds, heavy metals and other preservatives, the membrane should be rinsed or pre-dialyzed in ultra pure water or buffer for about 15 to 30 minutes before adding the sample. Special cleaning solutions may also be required for extremely sensitive applications and in cases when the presence of low level contaminants may interfere with downstream analysis of the sample.

However, you should not, in any case, boil the membrane since doing so can alter the pore rating and damage the membrane altogether. Also avoid touching the membrane with your bare hands since it can increase the risk of enzymatic and microbial contamination. To eliminate need of manipulating the membrane during setup and sample handling, improve sample recovery rate, and facilitate easy sample loading, consider using pre-assembled dialysis devices.

Please take note that dialysis membranes are designed for single use. They should not be re-used for the same protein samples since it may increase the likelihood of contamination. Keep in mind that handling and dialysis conditions (pH, temperature and chemical exposure) can also alter the integrity of the membrane and increase the risk of leaks.

Storage of Dialysis Membrane Tubing

Dialysis membranes have varying shelf life. Dry packaged dialysis membranes have a shelf-life of 5 years while wet packaged (0.05% sodium azide solution) membranes and irradiated membranes can be stored for 3 and 1.5 years, respectively. Do not allow membranes to dry out after wetting because it can decrease the pore size and increase the risk of leakage. In addition, you should not allow membranes to freeze since the ice crystals may rupture the membrane and cause it to leak. In either case, consider discarding the membrane.

Dialysis Procedure

There are several things that you should avoid when performing a dialysis procedure. Here are some of them.

• Do not perform dialysis against a low salt concentration buffer or dialyze against pure deionized water since this will cause the osmotic pressure to draw water into the tubing and rupture the membrane. For best results, perform a serial dialysis using buffers with decreasing concentration of solutes (salt) and try to reduce the order of magnitude of solute concentration by a factor of 10 to 1 at each buffer exchange.
• In cases where you need to return the sample to its original concentration, you may decrease the sample volume or add a solution of hygroscopic compound instead of ordinary dialysate. Just make sure that the hygroscopic compound is composed of molecules that are larger than the pore size of the dialysis tubing to prevent contamination.
• Do not tie knots in the dialysis tubing since these do not provide an effective seal against leakage. Use the appropriate dialysis closures to ensure safe dialysis of your sample. Standard regenerated cellulose (RC) membranes can be sealed using any of the standard dialysis tubing closures while cellulose ester (CE) membranes require the gentler Universal closure. Using a closure with a sealing width of 4 to 10 mm longer than the flat width of the dialysis tubing is highly recommended.

Image Source : Steve Davis