During the protein purification process, several issues need to be noted. Firstly, the pH value should be controlled throughout the purification process to avoid irreversible conformational changes in proteins due to excessive acidity or alkalinity. The buffer solution should have the appropriate pH value and be harmless to proteins. When purifying proteins from plants and fungus, the larger buffer capacity is usually required. At this time, attention should be paid to the concentration of the buffer solution, as well as whether the components of the buffer solution are appropriate and whether they will cause side reactions, etc. Proteins containing free sulfydryl groups (when the sulfydryl groups are not bound into disulfide bonds) are particularly prone to oxidation. Therefore, the reducing environment must be maintained. Some reducing agents containing sulfydryl groups can be added, and the entire system should be kept in the oxygen-free state to solve this problem. However, in some cases, the added sulfydryl compounds may inhibit the activity of the protein.

During the purification process, it is usually necessary to maintain the low temperature because there are proteolytic enzymes in the cells, which are activated after tissue homogenization and can degrade proteins. Therefore, the low temperature must be maintained to reduce the activity of proteolytic enzymes. However, it should be noted that sometimes low temperatures can also destroy the quaternary structure of some enzymes. Generally, proteins are relatively stable at 0°C, but acetone carboxylase is sensitive to cold and it is stable at 25°C. Some enzymes require -20°C or -70°C to maintain their activity.

One of the biggest problems in protein purification is that proteins are inevitably exposed to solution environments that are very different from their physiological states throughout the purification process. This is particularly significant for proteins whose functions are highly regulated within living cells. In contrast, some proteins, such as secretion proteins, can adapt to significant environmental changes without altering their structure or function. To reduce the spontaneous denaturation of proteins in dilute aqueous solutions, small-molecule substances (such as sucrose, glycerol, etc.) or other proteins (such as bovine serum albumin, gelatin, etc.) can be added to the protein solution to stabilize the proteins. This is one method that mimics the intracellular environment and can reduce the harmful effects of water. This is also the reason why albumin is added as a stabilizer in many gene protein drugs. Sometimes even dimethyl sulfoxide, dimethylformamide, etc. are added, and the concentration needs to be tested, generally ranging from 1% to 10%. A few proteins can maintain their activity in polar media with high ionic strength, such as KCl, NaCl, (NH4)2SO4, etc.

In addition, there are some commonly used methods that can effectively reduce protein denaturation. For example, the presence of some divalent ions (such as Mg2+, Ca2+, etc.) can help stabilize proteins or protein complexes; when purifying enzymes, specific substrates can be added; sometimes chelating agents (such as ethylenediaminetetraacetate) are used to remove unwanted ions, especially those harmful metal ions brought by water sources. These methods can be utilized depending on the specific situation.

Another factor to note is that even with great care during the purification process, proteins may undergo structural changes. Therefore, even if the activity of the purified protein is largely retained, its conformation may differ from the natural conformation or the conformation with complete function. In such cases, the properties of the protein measured in vitro may not accurately reflect the characteristics of these macromolecules.

Dissociation of proteins containing multiple subunits is one special type of denaturation. In the purification of proteins, when measured activity is expressed by only one kind of subunit, other subunits may be lost because they are not noticed. Some proteins have the multifunctional form in the body, and their biological activity is displayed by two or more proteins determined by different genes. Caution should be exercised when inferring the full biological function of the purified protein from its properties. It is useful to analyze the overall activity in the purification process. In the early stage of purification, if the total vitality is used as a benchmark, it is hoped that 50% of the total vitality can be recovered at the end of purification, but the actual yield can be achieved is often only 5%-20%. In some cases, especially when the large amount of raw material is available, we can concentrate on obtaining the high specific activity sample without worrying about the yield.