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.

Materials

• Dialysis membrane of desired molecular cut-off
• 10 mM sodium bicarbonate
• 10 mM Na2
• EDTA, pH 8.0
• 20% to 50% (v/v) ethanol

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

Microcentrifuge Dialysis

Health and Safety

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