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

Basically, there are two methods that are commonly used in the preparation of protein samples to make them suitable for long-term storage and compatible with downstream applications - dialysis and gel filtration. So, how do you know which one to use? Here are some things you need to consider if you want to increase your chances of making the right decision.

When doing a Western blot procedure, the macromolecules are first separated using gel electrophoresis. The separated macromolecules are transferred onto a second matrix (nitrocellulose or polyvinylidene difluoride (PVDF) membrane) and the membrane is blocked to prevent the nonspecific binding of antibodies to its surface. The transferred protein is then complexed with an enzyme-labeled antibody (probe) and an appropriate substrate is added to produce a detectable product.

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.

Hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state.  HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding. So, how does HIC work? What are the principles behind this technique? To understand the mechanism behind this type of chromatography, here are some things you definitely need to know.