Salting out
Encyclopedia
Salting out is a method of separating proteins based on the principle that proteins are less soluble at high salt
concentrations. The salt concentration needed for the protein to precipitate out of the solution
differs from protein to protein. This process is also used to concentrate dilute
solutions of proteins. Dialysis
can be used to remove the salt if needed.
s and hydrophilic amino acid
s in protein
molecules.
After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation
and allow proteins to form hydrogen bonds with the surrounding water molecules. If enough of the protein surface is hydrophilic, the protein can be dissolved in water.
When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein.
As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other. This process is known as salting out.
Unwanted proteins can be removed from a protein solution mixture by salting out as long as the solubility of the protein in various concentrations of salt solution is known.
After removing the precipitate by filtration
or centrifugation
, the desired protein can be precipitated by altering the salt concentration to the level at which the desired protein becomes insoluble.
Also some ions can denature certain proteins so if assays on the function of proteins are intended then either a different ion or an alternative purification method should be used.
In attempting to remove a product from water, NaCl is often used to increase the ionic strength of water, thereby increasing its polarity, and then the product is moved into the organic layer where it can be extracted.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988 February 11; 16(3): 1215.
Salt
In chemistry, salts are ionic compounds that result from the neutralization reaction of an acid and a base. They are composed of cations and anions so that the product is electrically neutral...
concentrations. The salt concentration needed for the protein to precipitate out of the solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
differs from protein to protein. This process is also used to concentrate dilute
Dilution
Dilution may refer to:* Reducing the concentration of a chemical* Serial dilution, a common way of going about this reduction of concentration* Homeopathic dilution* Dilution , an equation to calculate the rate a gas dilutes...
solutions of proteins. Dialysis
Dialysis (biochemistry)
In biochemistry, dialysis is the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing....
can be used to remove the salt if needed.
Principle
There are hydrophobic amino acidAmino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s and hydrophilic amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s in protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
molecules.
After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation
Solvation
Solvation, also sometimes called dissolution, is the process of attraction and association of molecules of a solvent with molecules or ions of a solute...
and allow proteins to form hydrogen bonds with the surrounding water molecules. If enough of the protein surface is hydrophilic, the protein can be dissolved in water.
When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein.
As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other. This process is known as salting out.
Application
As different proteins have different compositions of amino acids, different protein molecules precipitate at different concentrations of salt solution.Unwanted proteins can be removed from a protein solution mixture by salting out as long as the solubility of the protein in various concentrations of salt solution is known.
After removing the precipitate by filtration
Filtration
Filtration is commonly the mechanical or physical operation which is used for the separation of solids from fluids by interposing a medium through which only the fluid can pass...
or centrifugation
Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...
, the desired protein can be precipitated by altering the salt concentration to the level at which the desired protein becomes insoluble.
Caution
Certain ions can increase the solubility of a protein when the concentration of the ions increases, instead of decreasing the solubility of the protein.Also some ions can denature certain proteins so if assays on the function of proteins are intended then either a different ion or an alternative purification method should be used.
In attempting to remove a product from water, NaCl is often used to increase the ionic strength of water, thereby increasing its polarity, and then the product is moved into the organic layer where it can be extracted.
Reference
Physical biochemistry - SheehanMiller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988 February 11; 16(3): 1215.
See also
- Salting inSalting inSalting in refers to the effect where increasing the ionic strength of a solution increases the solubility of some solute . This effect tends to be observed at lower ionic strengths....
- Ammonium sulfate precipitationAmmonium sulfate precipitationAmmonium sulfate precipitation is a method used to purify proteins by altering their solubility. It is a specific case of a more general technique known as salting out....
- Hofmeister seriesHofmeister seriesThe Hofmeister series or lyotropic series is a classification of ions in order of their ability to salt out or salt in proteins. The effects of these changes were first worked out by Franz Hofmeister, who studied the effects of cations and anions on the solubility of proteins.Hofmeister discovered...