Ammonium sulfate precipitation
Encyclopedia
Ammonium 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
.
Ammonium sulfate
is commonly used as its solubility is so high that salt solutions with high ionic strength are allowed.
The solubility of proteins varies according to the ionic strength of the solution, and hence according to the salt concentration. Two distinct effects are observed: at low salt concentrations, the solubility of the protein increases with increasing salt concentration (i.e. increasing ionic strength), an effect termed salting in
. As the salt concentration (ionic strength) is increased further, the solubility of the protein begins to decrease. At sufficiently high ionic strength, the protein will be almost completely precipitated from the solution (salting out).
Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. The commonly used salt is ammonium sulfate, as it is very water soluble, forms two ions high in the Hofmeister series
, and has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to the required concentration, expressed as a percentage concentration of the saturated solution (a 100% solution).
In the preliminary test, the ammonium sulfate concentration is increased stepwise, and the precipitated protein is recovered at each stage. This is usually done by adding solid ammonium sulfate, but calculating how much ammonium sulfate to add to a solution at one concentration to achieve a desired higher concentration is tricky, since addition of ammonium sulfate significantly increases the volume of the solution. The amount to add can be determined either from published nomograms or by using an online calculator http://www.encorbio.com/protocols/AM-SO4.htm. Each protein precipitate is dissolved individually in fresh buffer and assayed for total protein content and amount of desired protein. The aim is to find the ammonium sulfate concentration which will precipitate the maximum proportion of undesired protein, whilst leaving most of the desired protein still in solution or vice versa.
The precipitated protein is then removed by centrifugation and then the ammonium sulfate concentration is increased to a value that will precipitate most of the protein of interest whilst leaving the maximum amount of protein contaminants still in solution. The precipitated protein of interest is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification.
This technique is useful to quickly remove large amounts of contaminant proteins, as a first step in many purification schemes. It is also often employed during the later stages of purification to concentrate protein from dilute solution following procedures such as gel filtration.
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
. It is a specific case of a more general technique known as salting out
Salting out
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...
.
Ammonium sulfate
Ammonium sulfate
Ammonium sulfate , 2SO4, is an inorganic salt with a number of commercial uses. The most common use is as a soil fertilizer. It contains 21% nitrogen as ammonium cations, and 24% sulfur as sulfate anions...
is commonly used as its solubility is so high that salt solutions with high ionic strength are allowed.
The solubility of proteins varies according to the ionic strength of the solution, and hence according to the salt concentration. Two distinct effects are observed: at low salt concentrations, the solubility of the protein increases with increasing salt concentration (i.e. increasing ionic strength), an effect termed salting in
Salting in
Salting 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....
. As the salt concentration (ionic strength) is increased further, the solubility of the protein begins to decrease. At sufficiently high ionic strength, the protein will be almost completely precipitated from the solution (salting out).
Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. The commonly used salt is ammonium sulfate, as it is very water soluble, forms two ions high in the Hofmeister series
Hofmeister series
The 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...
, and has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to the required concentration, expressed as a percentage concentration of the saturated solution (a 100% solution).
In the preliminary test, the ammonium sulfate concentration is increased stepwise, and the precipitated protein is recovered at each stage. This is usually done by adding solid ammonium sulfate, but calculating how much ammonium sulfate to add to a solution at one concentration to achieve a desired higher concentration is tricky, since addition of ammonium sulfate significantly increases the volume of the solution. The amount to add can be determined either from published nomograms or by using an online calculator http://www.encorbio.com/protocols/AM-SO4.htm. Each protein precipitate is dissolved individually in fresh buffer and assayed for total protein content and amount of desired protein. The aim is to find the ammonium sulfate concentration which will precipitate the maximum proportion of undesired protein, whilst leaving most of the desired protein still in solution or vice versa.
The precipitated protein is then removed by centrifugation and then the ammonium sulfate concentration is increased to a value that will precipitate most of the protein of interest whilst leaving the maximum amount of protein contaminants still in solution. The precipitated protein of interest is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification.
This technique is useful to quickly remove large amounts of contaminant proteins, as a first step in many purification schemes. It is also often employed during the later stages of purification to concentrate protein from dilute solution following procedures such as gel filtration.