What are the methods of precipitating proteins, and what are their own characteristics?

What are the methods for precipitating proteins? What are the characteristics of each?

Answer: (1) Salting out method, this method does not destroy the natural state of the protein, and the precipitated protein can be invariant, so the salting out method is to separate and prepare proteins or protein biological agents.

of common methods.

2) Organic solvent precipitation method, which precipitates proteins by destroying the hydration membrane of proteins, and this method can denature proteins at room temperature.

Low temperatures can slow down the rate of denaturation.

3) Heavy metal salt precipitation method, which can combine with protein to form water-insoluble protein salt precipitation, causing protein denaturation. Clinically, it is used to save heavy metal salt poisoning.

Salting-out refers to the phenomenon of adding neutral salts to the aqueous solution of proteins, and precipitating the proteins with the increase of salt concentration. Neutral salt is a strong electrolyte, and the solubility is large, in the protein solution, on the one hand, it competes with the protein for water molecules and destroys the water film on the surface of the protein colloidal particles; On the other hand, a large amount of neutralization of the charge on the protein particles, so that the protein particles in the water accumulate and precipitate, as follows:

1. Destruction of the hydration layer.

In a high concentration of neutral salt solution, because the salt ions are more hydrophilic than proteins, they compete with protein colloids to bind to water, destroying the hydration layer of proteins. In a high-concentration neutral salt solution, because the protein and salt ions are attractive to the water molecules in the solution, the phenomenon of hydration is produced, but there is a competition between them, when a large number of neutral salts are added, the ions produced by salt dissociation compete for most of the free water in the solution, thereby destroying the hydration of the protein, causing the protein solubility to decrease, so it is precipitated from the solution.

2. Destroyed the charge.

Since salt is a strong electrolyte with strong dissociation effect, the dissociation of salt can inhibit the dissociation of weak electrolytes of proteins, so that the charge of proteins is reduced, and it is easier to aggregate and precipitate.

Protein precipitation The stability of proteins in solution is conditional and relative. If the conditions change and the stability of the protein solution is destabilized, the protein molecules aggregate into large particles and precipitate.

The stability of a protein solution is related to particle size, charge, and hydration, and as long as these conditions are disrupted, the protein will naturally precipitate out of the solution.

There are the following methods for precipitating proteins:

1. Salting-out method.

2. Organic solvent precipitation method.

3. Heavy metal salt precipitation method.

4. Alkaloid reagent and certain acids (trichloroacetic acid, sulfosalicylic acid, nitric acid, etc.) precipitation method.

5. Heating denaturation precipitation method.

Protein denaturation: A phenomenon in which the natural conformation of a protein is disrupted, resulting in the loss of its biological activity. When the protein is subjected to light, heat, organic solvents and some denaturants, the secondary bonds are broken, resulting in the destruction of the natural conformation and the loss of the biological activity of the protein.

The phenomenon of protein molecule condensation and precipitation from the solution is called protein precipitation, denatured proteins are generally easy to precipitate, but they can also be unchanged and make proteins precipitate, and under certain conditions, denatured proteins can also not precipitate.

The hydrocolloidal particles formed by proteins have two stabilizing factors, namely the hydration layer and the electrical charge on the surface of the particles. If there are no additional conditions, they will not agglutinate with each other. However, without these two stabilizing factors (e.g., adjusting the pH of the solution to the isoelectric point and adding a dehydrating agent), proteins tend to agglutinate and precipitate.

It can be seen that if the pH of the protein solution is adjusted to the isoelectric point, the protein molecule is in an isoelectric state, although the intermolecular repulsion of the same charge disappears. However, there is also a hydration film to play a protective role, generally not to cause coagulation, if a certain dehydrating agent is added at this time, remove the hydration membrane of protein molecules, then the protein molecules will coagulate with each other and precipitate; Conversely, if the protein is dehydrated first, and then the pH is adjusted to the isoelectric point, the protein can also be precipitated.

The reversible precipitation of proteins generally occurs at the time of salting-out, that is, a concentrated salt solution is added to the protein solution to allow the protein to be precipitated, in which case the spatial conformation of the protein is still intact, and the reconstituted protein is still biologically active. The irreversible precipitation of proteins is generally considered to be protein denaturation, that is, the specific spatial conformation of the protein is destroyed under the action of certain physical and chemical factors, resulting in the loss of its biological activity. Reversible precipitation of proteins is mostly used for extraction and purification, such as the preparation of casein from milk by salting out method.

Irreversible precipitation of proteins can be used for sterilization, sterilization, e.g. high-temperature sterilization of medical devices.

Hello landlord, there are two kinds of protein precipitation you said, after adding light metal salts (such as sodium chloride), it will coagulate, but it is reversible, and it becomes clear after adding enough water. The other is that after heating, adding heavy metal salts (such as lead acetate), concentrated acid, or adding formaldehyde, ethanol and other organic substances, it will denature and produce solid precipitation, which is irreversible.

The details are as follows: 1. Salting-out: adding high-concentration neutral salts to the protein solution destroys the stability of the colloid, neutralizes the charge of the protein, and can destroy the hydration membrane. The precipitated protein by this method is generally unchanged, and the neutral salt can be removed by dialysis, and the biological activity can still be maintained.

2. Organic solvent sales: use their strong hydrophilicity to destroy the hydration membrane, because they can not neutralize the charge of the protein, it is necessary to precipitate the protein near the pi. At low temperatures, denaturation occurs slowly and can be used to prepare plasma proteins. It will denature at room temperature.

3. Heavy metal salts: negatively charged proteins combine with positively charged heavy metal ions to form insoluble salts and precipitate. When using, it is necessary to adjust the pH so that it is greater than the PI, and denaturation will generally occur.

4. Alkaloid reagents and certain acids: positively charged proteins can form salts and precipitate with the acid segments of alkaloid reagents (such as picric acid, tannic acid, tungstic acid) or the acid roots of some acids (such as trichloroacetic acid, nitric acid, perchloric acid). Degeneration usually occurs.

It is clinically used for blood chemistry analysis, and is commonly used to remove proteins from the blood.

The principle of protein precipitation by salting out is to reduce the solubility of protein.

Proteins are coagulated and precipitated from solution.

Protein precipitation (protein

precipitation), precipitation is the process by which the solute in the solution changes from the liquid phase to the solid phase. The phenomenon of protein precipitation from solution is called protein precipitation. Commonly used methods for protein precipitation are salting-out and isoelectric point.

precipitation, precipitation of organic solvents, alkaloid reagents, and certain acids (e.g., trichloroacetic acid.

precipitation, etc.