Accurately studying proteins is often not possible without first conducting a process called protein purification. Protein purification removes impurities, contaminants, and coexisting proteins until all that is left is the protein the researcher wishes to study. Purification makes the results of protein studies more accurate by eliminating potential variables. One of the most common processes used for the purification of proteins is dialysis.
What Is Protein Dialysis?
Protein dialysis is a laboratory-scale process that purifies a protein by removing or reducing salt concentration. It separates small and large molecules through the process of molecular diffusion. Diffusion allows small molecules to pass through a permeable membrane but stops larger molecules from going through. Dialysis uses diffusion to separate smaller salt molecules from larger non-salt molecules. Dialysis can also separate contaminants such as reducing agents, labeling reagents, and preservatives.
When working with protein samples, dialysis is often an important part of protein stability analysis. Measuring the stability of a protein precisely usually takes purifying the sample first – although some modern technologies can accurately gauge protein stability with non-prepped samples. Accurate protein research usually requires protein purification through dialysis and/or other methods to remove contaminants before further analyzing the protein’s functions, interactions, and stability.
How Does Dialysis Work?
Protein dialysis uses the thermal movement of molecules in a special solution to separate them into areas of higher or lower concentration, until reaching equilibrium. During dialysis, the smaller, unwanted molecules will fall through a semipermeable membrane into a second chamber, usually filled with liquid or dialysate. The larger molecules that are too big to pass through the membrane will stay in the original sample chamber. This effectively reduces the quantity of small molecules within a protein sample.
Dialysis relies on heat to diffuse a molecule. Increasing the temperature of dialysis, therefore, increases the rate of diffusion. Choosing the correct temperature for your dialysis takes understanding the thermal stability of the molecule you are trying to study. The concentration of the molecule will also determine the rate of diffusion. As the weight of a molecule increases, the rate of diffusion decreases. Finally, the rate of dialysis is proportional to the membrane’s surface area, and inversely proportional to its width. Laboratory dialysis applications typically have a thickness to provide a good diffusion rate.
Benefits of Protein Dialysis
The most important reason to use protein dialysis is to separate the protein you wish to study from contaminants. However, dialysis also acts as a buffer exchange. This is the process of transporting the protein sample to a different buffer system for a downstream application, such as affinity chromatography. Protein dialysis makes buffer exchange possible by separating and desalting the protein, enabling the researcher to recover molecules of interest. After dialysis, the larger molecules emerging from the column will remain in the equilibration buffer. The original buffer remains in the resin – thus completing buffer exchange.
Protein dialysis typically uses a sealed dialysis membrane, along with a selected buffer, or liquid. The buffer you choose can determine the results of your dialysis. Dialysis against Aquacide 11A, for example, removes water through the dialysis tubing. Immersible-CX Ultrafilters remove everything below a specific molecular weight cutoff. You may also use a centrifugal concentrator, depending on your goal.