How dialysis is used in protein research?
In a nutshell, dialysis is a common laboratory technique used in removing contaminants in a solution by selective and passive diffusion through a semi-permeable membrane such as dialysis tubing. In life science research, dialysis is most commonly used in removing small, unwanted molecules such as salts, reducing agents (e.g. dithiothreitol or DTT and 2-mercaptoethanol or BME), preservatives (e.g. sodium azide and thimerosol) and non-reacted crosslinking or labeling reagents (e.g. sulfo-SMCC and biotin) in a solution. This technique is also quite useful for buffer exchange and drug binding studies.
Dialysis works by diffusion. By definition, diffusion is the random, thermal movement of molecules in a solution from areas of higher concentration to areas of lower concentration until equilibrium is reached. Differential diffusion patterns are achieved by separating the sample and the buffer solution (dialysate) with a semi-permeable membrane to separate molecules in the sample and dialysate.
Since the larger molecules cannot pass through the pores of the membrane, they will stay in the sample chamber while the small molecules will easily diffuse across the membrane. In time, the overall concentration of the molecules in the sample and the dialysate will change and equilibrium will be reached. By dialyzing your protein sample, you can remove the small molecules that have effectively passed through the membrane. You can also decrease the concentration of contaminants with each buffer change and prevent them from interfering with the subsequent steps in the experimental procedure.
General Dialysis Protocol
Dialysis is a relatively straightforward process. All you need is your protein sample and dialysate buffer, a dialysis membrane in appropriate format and molecular weight cut-off (MWCO), and a container to hold the buffer. Dialysis membranes are usually made of a film of regenerated cellulose or cellulose esters and have MWCOs ranging from ~1,000 to ~50,000kDa.
It is interesting to note that there is no universal procedure that will suit all applications. The volume of the sample, the size of the molecules being separated, the type and geometry of the membrane used, and the subjective nature of the dialysis endpoint should all be taken into account to optimize the results that you will get from the procedure.
However, there is a general guideline that you can follow to get the results that you need. Here is a typical dialysis procedure that you can follow to remove unwanted molecules from your protein samples.
Prepare the membrane according to instructions.
Load the sample into dialysis tubing, cassette or device and dialyze for 2 hours. You can perform this step at room temperature or 4°C.
Change the dialysis buffer and dialyze for another 2 hours.
Change the dialysis buffer and dialyze overnight at 4oC.
Please note that two factors play a vital role in creating and maintaining the concentration-differential across the membrane – (1) the difference in the composition of the sample and the dialysis buffer solutions, and (2) a high buffer-to-sample volume-ratio. The number of buffer changes and the dialysis time also affect the outcome achieved in dialysis.
Factors Affecting Performance
The time required to complete the procedure is dependent on the same factors that determine the rate of diffusion of molecules. As such, it can be affected by the following:
Temperature. Since increasing the temperature speeds up the rate of diffusion, the procedure will proceed faster at room temperature than at 4oC. However, you will need to take the thermal stability of the molecule of interest into account when determining the optimal temperature.
Concentration and molecular weight of the molecule. The higher the concentration, the faster the rate of diffusion. However, as the molecular weight increases, the rate of movement slows down considerably.
Surface area and thickness of the membrane. The diffusion rate is directly proportional to the surface area of the membrane and is inversely proportional to its thickness.
By properly dialyzing your protein samples, you can remove all the unwanted molecules and get accurate results from your experiment.