The Protein Man's Blog | A Discussion of Protein Research

In biochemistry, dialysis refers to the process of separating small, unwanted compounds from macromolecules in the sample solution through selective and passive diffusion. Basically, the sample and the buffer solution (dialysate) are placed on opposite sides of the membrane. Since dialysis works by diffusion, molecules will naturally move from areas of higher concentration to areas of lower concentration until the point where equilibrium is reached. This, in turn, will facilitate the separation of molecules in both the sample and the dialysate.

Since large molecules cannot pass through the pores of the membrane, they will be retained on the sample side of the chamber. However, the unwanted molecules (including buffer salts (Tris and PBS), reducing agents (DTT, BME), preservatives such as sodium azide and thimerosol and/or non-reacted crosslinking or labeling reagents such as sulfo-SMCC and biotin), which are significantly smaller in size, can easily diffuse through the semi-permeable membrane into the dialysate. This reduces the concentration of these molecules in the sample until equilibrium is obtained across the entire solution.

Generally, the rate of dialysis slows as it approaches equilibrium so you need to change the dialysate after several hours to re-establish the concentration differential that powers the dialysis process. By allowing dialysis to proceed to equilibrium before changing the dialysate buffer, you will be able to purify the substances retained by the membrane by a factor equal to the ratio of buffer volume to sample volume.

Dialysis Procedure

Due to the fact that there are a number of variables associated with each sample and that the completion point of the process is somewhat subjective, there is no universal dialysis procedure that will suit all applications. Keeping this in mind, here is a typical dialysis procedure that you can use for your protein samples.

1. Pre-wet or prepare the membrane according to manufacturer's instructions.
2. Load the sample into dialysis tubing or device.
3. Dialyze for one to two hours at room temperature.
4. Change the dialysis buffer and dialyze for another hour or two.
5. Change the dialysis buffer and dialyze overnight at 4°C.

The difference in the composition between the sample and the dialysate creates a concentration differential across the membrane and this powers the entire process. As such, you should use a high buffer-to-sample volume-ratio to maintain the concentration gradient. For best results, make sure that your dialysate is about 200 to 500 times larger than the sample volume. The number of dialysate buffer changes and the dialysis time will also affect your results.

Improving Dialysis Performance

Basically, you can improve the rate and efficiency of your experiment by controlling the factors affecting diffusion.

• Heat. Heat affects the thermodynamics of molecules so you can speed up diffusion and dialysis by increasing the temperature. However, please take note that the thermal stability of your protein of interest is more important than the rate by which your experiment will proceed.
• Concentration. The rate of diffusion is directly proportional to the concentration of a molecule. As a result, the probability that the molecules will come into contact with the dialysis membrane and diffuse through the other side will be higher if there is high concentration of these molecules.
• Molecular weight. The rate of diffusion is inversely proportional to the molecular weight (MW) of a molecule so the higher the MW, the slower the rate of movement and the lower the chance of diffusion through the membrane.
• Membrane. The rate of dialysis is directly proportional to the surface area of the membrane and inversely proportional to its thickness. As such, choose a membrane that provides the maximum surface area-to-volume ratio.  Since the thickness of the membrane also determines the efficiency of the procedure, use one that is about 0.5 to 1.2 mil (12 to 30µm) thick to ensure good diffusion rate and structural integrity.
• Stirring. This efficiently breaks up the macro-environment outside the Nernst layer and helps maintain the concentration differential needed to propel the diffusion process to completion.
• The addition of chemical sorbents to the dialysate. There are some sorbents that, when added to the dialysis buffer, can shift the equilibrium of the solution. This helps to remove even more of the small molecules that may be present and results in a cleaner sample.

To further improve the efficiency of your dialysis procedure, you should consider using a dialysis device that ensures 100% recovery (even if precipitation occurs) to prevent the loss of your precious samples. In addition, when using proprietary products to enhance the rate of your experiment, consider using those that do not contain any chemicals that will interfere or modify your reagents, or cross the dialysis membrane.