Precipitation Techniques

The theory behind such techniques is complex and not clearly understood, but the techniques themselves are straightforward. To say that an increase in protein-protein interactions leads to precipitation whereas protein-solvent interaction favors solubility is a simplistic but nonetheless useful paradigm. Precipitation techniques do not, by themselves, achieve a great increase in the purity of a protein solution, but generally they result in an increase in concentration and have a role to play in many protein purification protocols.

The most common protein precipitation technique involves the use of ammonium sulphate, which has been the subject of a recent review (3). The widespread use of ammonium sulfate can be ascribed to the fact that it is very soluble (saturated solutions have a concentration in the region of 4 M), the density of solutions do not compromise collection of precipitates by centrifugation and its use does not promote denaturation of proteins. The addition of ammonium sulfate will cause a neutralization of the surface charge of the protein and a decrease in the effective concentration of water leading to a decrease in protein solvent interactions.

Polyethylene glycol also has a long history of use as an agent for protein precipitation (4). It shares some of the positive attributes of ammonium sulphate in having a low heat of solution and not promoting denaturation of proteins. It appears that after the addition of polyethylene glycol, proteins are excluded from the space occupied by the hydrated polymer, and their effective concentration is increased to a level incompatible with solubility. It is less effective in the purification of IgG but is useful for the isolation of the larger IgM.

A number of other precipitating agents have been used in the purification of proteins. In some instances, the use of sodium sulfate can result in a purer antibody preparation, but generally it does not offer advantages over ammonium sulphate. Caprylic (octanoic) acid, however, offers a different approach and also has a long history of use (5). Conditions can be created where this short chain fatty acid will effectively precipitate the majority of serum proteins with the exception of the immunoglobulins.

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