Ammonium sulfate precipitation

Ammonium sulfate precipitation is a method of protein purification by altering solubility of protein. It is a specific case of a more general technique known as salting out.

Additional recommended knowledge

Correct Test Weight Handling Guide: 12 Practical Tips

Guide to balance cleaning: 8 simple steps

How to quickly check pipettes?

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 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 on line calculator [1]. 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.

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.