Why is saturated ammonium sulfate used in the salting out experiment of proteins

Metal cations in salting out agents.

The greater the number of charges, the stronger the salting out. In the case of the same charge number of cations, salting out is inversely proportional to the radius of the cation, because the cations with high valence and small radius have a stronger hydration ability, so the free water molecules are made.

The effect of the reduction is greater.

Since saturated ammonium sulfate is a strong electrolyte.

The dissociation effect is strong, and the dissociation of saturated ammonium sulfate 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.

Because the local salt concentration is too high, it may cause irreversible denaturing precipitation. This part of the precipitate cannot be reconstituted.

If a protein solution is added to the ammonium sulfate solution, the protein solution added first will inevitably be denatured due to the high salt concentration.

Therefore, it is necessary to slowly add ammonium sulfate solution to the protein solution and stir gently. Add ammonium sulfate drop by drop.

Because the serum protein exists in the solution, more or less precipitated will carry a part of the water, and it cannot be a simple serum protein, so it is a solution. Salting out generally refers to the process of adding inorganic salts to the solution to reduce the solubility of a substance and precipitate. Such as:

The process by which the protein is coagulated by the enrichment of (NH4)2SO4; The process of adding saturated sodium carbonate solution to the esterification reaction of acetic acid to ethyl acetate reduces the solubility of ethyl acetate and makes its stratification more obvious. After adding some inorganic salt solutions to some protein solutions, the solubility of the protein can be reduced, so that the protein can be coagulated and precipitated from the solution, which is called salting out, which is a physical change and can be restored. Adding certain heavy metal salts to some protein solutions can change the properties of the protein and condense, and then precipitate from the solution, this effect is called denaturation, property change, is a chemical reaction, and cannot be recovered.

Animal fat or vegetable oil and sodium hydroxide are stirred and heated in a saponification pot in a certain proportion, and the high-grade fatty acid sodium, glycerol, and water formed after the reaction form a mixture (colloid). Add salt particles to the pot, stir and let stand to separate the sodium of advanced fatty acids from glycerin and water, and float on the liquid surface. (This reaction is used to make soap) simply means that the protein is precipitated by using a high concentration of neutral salt; The solubility (s) of the protein is different, and the concentration of salt used for precipitation is different.

Principle of salting out crystallization: salting out crystallization refers to the addition of a certain electrolyte salting out agent in the salt solution system, the added salting out agent, the hydration of its ions is stronger than other salts in the original solution, it reduces the number of free water molecules in the solution, so as to increase the effective concentration of the substances to be crystallized in the solution, so that the substances to be crystallized are crystallized and precipitated in the solution, which is salting out crystallization.

Because it has a strong salting out ability, it has a high solubility in water and a relatively low temperature coefficient of dissolution.

with ammonium sulfate. Protein purification by precipitation uses solid ammonium sulfate instead of ammonium sulfate solution because solid ammonium sulfate is much more efficient than ammonium sulfate solution.

The total ammonium sulfate precipitation method used to purify the protein by salting out method. When the ammonium sulfate solution reaches saturation, the protein precipitates in large quantities. Ammonium sulfate is used, because it does not contain water, and saturation can be reached very quickly.

This results in protein precipitation. The use of ammonium sulfate solution will bring in a large amount of water, even if the protein can be precipitated, because the ammonium sulfate solution brings in a large amount of water, will also lead to a large amount of dilution of the solution, so that the precipitation efficiency is greatly reduced. Therefore, it is necessary to use anhydrous solid ammonium chloride.

as a precipitating agent.

It is best to use solid ammonium sulfate to purify proteins by ammonium sulfate precipitation method, rather than using the principle of salting out of proteins actually used by ammonium sulfate precipitation method in ammonium sulfate solution, only when the ammonium sulfate concentration is high enough, the protein can be precipitated, and the use of ammonium sulfate solids can quickly reach the concentration of protein precipitation, which is conducive to protein purification.

Ammonium sulfate precipitation can be used to concentrate and partially purify proteins from a large number of crude formulations. This method allows the primary immunosphere to be separated from the sample and is a common method for immunoglobulin isolation.

Salting-out. It is a protein separation technology that causes the protein to precipitate by adding a high concentration of neutral salts (such as ammonium sulfate) to the protein solution. Not only most of the water in the original solution is converted into hydrated water of salt ions, but also the hydration layer on the surface of protein molecules combined by electrostatic action is also removed to solvate salt ions, so that the hydrophobic residues on the surface of protein molecules are fully exposed, so that aggregation and precipitation occur.

Salt dissolution refers to the addition of salt to make the substance soluble.

Increase. NH4) The advantages of SO4 as a precipitant are: because it is a divalent ionic neutral salt, and it has a high solubility in water, and the temperature coefficient of solubility is also low, it can exist at a high concentration at low temperature (4 °C) to compete for the water in the solution and the hydrated water on the surface of the protein branch, so that the protein solubility is effectively reduced, so that the protein can be precipitated out of the solution while maintaining activity.

The higher the charge number of the metal cation in the salting out agent, the stronger the salting out effect. In the case of the same charge number of cations, salting out is inversely proportional to the radius of the cation, because the cation with a high valence state and a small radius has a stronger hydration ability, so the effect of reducing free water molecules is greater.

Since saturated ammonium sulfate is a strong electrolyte with strong dissociation effect, the dissociation of saturated ammonium sulfate 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.

Summary. Hello, I am inquiring for you here, please wait a while, I will reply to you right away When doing bovine serum albumin experiments, what is the saturation of ammonium sulfate during salting-out? What kind of protein is precipitated?

Hello, I am inquiring for you here, please wait a while, I will reply to you immediately Hello, I am happy to answer for you. Utilize the principle of similarity dissolution. Proteins are organic substances that are easily soluble in organic solvents.

Ammonium sulfate is an inorganic solvent, so adding ammonium sulfate to the protein solution is quite envious of turning into ammonium sulfate solution, and the protein has a small solubility in the ammonium sulfate solution, which is quite precipitated from the saturated solution.

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Salting out is a protein separation technique in which a high concentration of neutral salts, such as ammonium sulfate, is added to a protein solution, causing the protein to precipitate. The salting out effect is mainly due to the addition of a large number of neutral salts to reduce the activity of water, not only most of the water in the original solution is converted into hydrated water of salt ions, but also the hydration layer on the surface of protein molecules combined by electrostatic action is also removed to solvate salt ions, so that the hydrophobic residues on the surface of protein molecules are fully exposed, so that aggregation and precipitation occur. Salt dissolution refers to the addition of salt to make the substance soluble.

Increase. Salt soluble.

In the aqueous solution of protein, a small amount of neutral salt [i.e., dilute concentration], such as ammonium sulfate, sodium sulfate, is added.

Sodium chloride, etc., will increase the charge on the surface of protein molecules, and strengthen protein molecules and water molecules.

so that the solubility of the protein in the aqueous solution increases. This phenomenon is called salt dissolution.

The dilute concentration can promote the dissolution of the protein in the lulu cover, which is called salt solubilization. At the same time, the dilute salt solution has the advantage of protecting the protein from being denatured due to the partial combination of salt ions and proteins, so a small amount of neutral salts such as NaCl is added to the extract, and the concentration is generally in molar liters. Phosphate is often used in buffers.

and carbonate isotonic salt solutions.

NH4) The advantages of SO4 as a precipitant are: because it is a divalent ionic neutral salt, and it has a high solubility in water, and the temperature coefficient of solubility is also low, it can exist at a high concentration at low temperature (4 °C) to compete for water in the solution and hydrated water on the surface of protein molecules, so that the protein solubility is effectively reduced, so that the protein can be precipitated from the solution while maintaining its activity.

Summary. I'm glad to answer for you: Saturation is the precipitation of white balls and semi-saturated spheres.

In serum protein salting-out, why is the globulin solution precipitated after adding saturated ammonium sulfate solution?

I'm glad to answer for you: Saturation is the precipitation of white balls and semi-saturated spheres.