Ammonium sulfate precipitation

Ammonium Sulfate Precipitation

Ammonium sulfate precipitation is a method used in biochemistry to purify proteins by altering their solubility. This technique exploits the fact that the solubility of proteins varies with the ionic strength of the solution. By adding ammonium sulfate, a highly soluble salt, the ionic strength of the solution is increased, leading to the selective precipitation of proteins.

Principle

The principle behind ammonium sulfate precipitation is based on the "salting out" effect. Proteins are less soluble in solutions of high ionic strength because the salt ions compete with the protein molecules for water molecules, which are necessary to keep the proteins in solution. As the concentration of ammonium sulfate increases, proteins begin to precipitate out of the solution at different rates, depending on their solubility.

Procedure

1. Preparation of the Sample: The protein solution is first clarified by centrifugation to remove any insoluble material. 2. Addition of Ammonium Sulfate: Ammonium sulfate is gradually added to the protein solution. This can be done by adding solid ammonium sulfate directly or by adding a saturated ammonium sulfate solution. 3. Precipitation: As the concentration of ammonium sulfate increases, proteins begin to precipitate. The process is usually carried out at low temperatures to prevent protein denaturation. 4. Collection of Precipitate: The precipitated proteins are collected by centrifugation. 5. Dissolution and Dialysis: The protein precipitate is dissolved in a buffer of lower ionic strength, and dialysis is performed to remove excess ammonium sulfate.

Applications

Ammonium sulfate precipitation is widely used in the initial stages of protein purification. It is a simple and cost-effective method to concentrate proteins and remove contaminants. This technique is often followed by more specific purification methods such as chromatography.

Advantages and Disadvantages

Advantages

• Cost-effective: Ammonium sulfate is inexpensive and readily available.
• Scalable: The method can be easily scaled up for large volumes.
• Stability: Proteins are generally stable in high salt concentrations, reducing the risk of denaturation.

Disadvantages

• Non-specific: It does not provide high specificity in protein purification.
• Labor-intensive: Requires careful control of conditions and multiple steps.

Also see

• Protein purification
• Chromatography
• Ionic strength
• Salting out