Solubility

• Some proteins are more water-soluble than others. If a protein has more hydrophilic ("water-loving") regions, it will be more soluble than one that is more hydrophobic ("water-fearing"). As the salt concentration increases, the less soluble and more hydrophobic proteins will precipitate first. Closely related proteins can't be separated by salting out, because they will precipitate at roughly the same salt concentration, but proteins that have very different structures and characteristics can often be separated in this way.
  
  

Salts

• Ammonium sulfate is the most popular salt for this procedure, because it has very high solubility in water even at very low temperature. Typically you want to keep your solution cold to inhibit the activity of proteases, proteins that chop up other proteins. The salt concentration is usually described by percentage of saturation, where 25 percent is 25 percent of saturation (the concentration at which no more salt can dissolve).
  
  

Centrifugation

• Typically, the salt is added gradually to the protein solution while stirring it rapidly (a magnetic stirrer is very useful for this kind of procedure). The solution is centrifuged, causing some of the proteins to precipitate. Often this procedure will be divided into several steps. You might, for example, start out with 25 percent of saturation (a 25 percent cut), then go to a 75 percent cut. This process allows you to divide the proteins into three groups -- the ones that precipitate at 25 percent, the ones that precipitate at 75 percent and the ones that precipitate in between.
  
  

Considerations

• Salting out will entail some loss of the protein you want to isolate. Each protein has its own solubility curve -- the solubility of the protein as a function of solute concentration ("ionic strength"). Consequently, even if the increased salt concentration is insufficient to precipitate all of the protein of interest, it may still precipitate some of it. Moreover, you cannot use salting out to isolate a specific protein, because other proteins will precipitate along with it. Typically, salting out is just the first step in a protein purification process.