Dialysis of Proteins

Theory

Dialysis is an alternative time-consuming but efficient alternative method for concentrating proteins and remove contaminating salts and small particles like urea from eluted proteins in preparation for a downstream process.

The process works by ensuring a concentration gradient with respect to your protein sample which is held in a semi-permeable membrane which allows exchange of molecules below a certain molecular weight given by Daltons (Da). The protein sample is placed within the tubing leaving sufficient room for expansion and the ends of the tubing closed by tying a know or using clips. The sample is then submerged into a solution whose composition varies greatly depending on what you wish to get rid of and by how much for example if I wanted to get rid of all the urea in my samples then in my outside solution I would have no urea in it. A concentration gradient is established and urea in the sample will diffuse through the membrane into the surrounding solution in order to achieve equilibrium. The amount of reduction is related to the volume of the surrounding solution and composition

Practice

In practice the exact procedure for dialysis varies hugely depending on the desired levels of purity vs sample loss. The protocol is only a guideline and one should modify it depending on their own needs

Preparation of Dialysis Tubing

In general, dialysis membranes need to be pre-treated before use for efficient dialysis to occur. Do check with manufacturer for protocols specific to that membrane. Below outlines a generic method for preparing membranes most of which are made of cellulose.

1. Remove membrane from the roll and cut into usable lengths (usually 8 to 12 in.). Always use gloves to handle dialysis membrane, as it is susceptible to a number of cellulolytic microorganisms. Membrane is available as sheets or preformed tubing.
2. Wet membrane and boil it for several minutes in a large excess of 10 mM sodium bicarbonate. (Recommended to conduct the boiling in a fume cupboard due to smell from the beaker)
3. Boil several minutes in 10 mM Na₂EDTA. Repeat. (Boiling speeds up the treatment process but is not necessary. A 30-min soak with some agitation can substitute for the boiling step.)
4. Wash several times in distilled water.
5. Store at 4°C in 20% to 50% ethanol to prevent growth of cellulolytic microorganisms. Alternatively, bacteriostatic agents (e.g., sodium azide or sodium cacodylate) may be used for storage; however, ethanol is preferred for ease and convenience.

Membrane Dialysis

1. Remove dialysis membrane from ethanol storage solution and rinse with distilled water. Secure clamp to one end of the membrane or knot one end with double-knots.
2. Fill membrane with water or buffer, hold the unclamped end closed, and squeeze membrane. A fine spray of liquid indicates a pinhole in the membrane; discard and try a new membrane.
3. Replace the water or buffer in dialysis membrane with the macromolecule-containing sample and clamp the open end. Again, squeeze to check the integrity of the membrane and clamps.(If dialyzing a concentrated or high-salt sample, leave some space in the clamped membrane; there will be a net flow of water into the sample, and if sufficient pressure builds up the membrane can burst.)
4. Immerse dialysis membrane in a beaker or flask containing a large volume (relative to the sample) of the desired buffer. Dialyze at least 3 hr at the desired temperature with gentle stirring of the buffer.
5. Change dialysis buffer as necessary.
6. Remove dialysis membrane from the buffer. Hold the membrane vertically and remove excess buffer trapped in end of membrane outside upper clamp. Release upper clamp and remove the sample with a Pasteur pipet.

Microcentrifuge Dialysis

For small volumes membrane dialysis will cause large sample losses, instead one can implement a microcentrifuge dialysis in 0.5μl or 1.5μl microcentrifuges depending on your requirements.

1. Puncture the lid of each microcentrifuge tube using a heated glass Pasteur pipet (wideend heated) or cork borer to completely remove the center part of the lid. Open lid and place a single layer of dialysis tubing over top of tube, then close lid to hold dialysis tubing in place.
2. Turn tube upside down and shake reaction mixture onto the membrane surface. Tape each tube, dialysis surface down, to the side of a beaker, then fill the beaker with your buffer e.g. PBS. Dialyze 2 hr at 4°C with stirring.
3. To recover the sample, remove microcentrifuge tube from the buffer and centrifuge briefly right-side-up.

General Notes

• We found that microcentrifuge dialysis is not as effective as with membrane dialysis due to the much smaller contact area however you do get much less in the way of sample loss. It would be recommended to change buffers a number of times for microcentrifuge dialysis
• Note that it is more effective to dialyze in lots of smaller volumes than one large volume as the reduction in concentration e.g. if dialyzing with 10μl within 100μl buffer then you will expect a 10-fold dilution and subsequently again if you change the buffer which is more effective than dialyzing in 200μl and not changing.

Health and Safety

• Always handle dialysis membranes with gloves because the membrane is susceptible to cellulolytic microorganisms.
• The largest cause for concern is the larger volumes you are working with therefore dialyze on a stable surface, consider dialyzing in cold room as opposed to the refrigerator to help minimise spillages.

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