Protein-precipitation agents

More detailed data on protein precipitation agents and their efficacy has been included in a recent review by Flanagan and coworkers158 and is presented in Table 1.17. 

ITABLE 4.12. Some protein-precipitation agents and their mechanisms of action... 

Buffer component protein-precipitating agent, excipient for pharmaceutical formulation. 

Typicai protein precipitation agents are saits (e.g., ammonium suifate), PEG (e.g., PEG 20,000), poiyeiectroiytes (e.g., carboxymethyi ceiiuiose), and organic soivents (e.g., acetone). 

Samples with higher protein levels (yogurts), are initially treated with hydrochloric acid and after protein precipitation the supernatant is filtered and injected into the HPLC column. The separations performed with a LiChroCART RP18 column used a mixture of acetonitrile and formic acid as the mobile phase. A baseline quantification of the carminic acid was possible in the presence of other coloring agents, with excellent recuperation, selectivity, accuracy, and precision. ... 

The stain/fixation method is usually used for surface markers that can withstand fixation and is followed by the application of a DNA-binding fluoro-chrome. The fixation/stain method is used not only for surface markers that can withstand fixation, but also for intracellular constituents, such as cytoplasmic proteins, nuclear membrane, and nuclear proteins. This is accomplished by using a crosslinking fixative (e.g., paraformaldehyde [PFA] or formalin) followed by a permeabilizing agent (e.g., Triton X-100, Tween-20, saponin, or lysolecithin). Some of the precipitating agents (e.g., ethanol, methanol, or acetone) can also be used for permeabilization after the initial fixation with PFA or formalin, or they can be used alone for both fixation and permeabilization (see Chapter 8). 

Pasteur pipette with rubber squeezer Protein solution Precipitating agents... 

Organic solvents also decrease protein solubility, but they are not as widely used as ammonium sulfate. They are thought to function as precipitating agents in two ways (1) by dehydrating proteins, much as ammonium sulfate does, and (2) by decreasing the dielectric constant of the solution. The organic solvents used (which, of course, must be miscible with water) include methanol, ethanol, and acetone. 

A note of caution is that the precipitating agents just mentioned all result in fairly complete precipitation for large (intact) proteins only. Remember that these deproteinization studies usually involve plasma proteins. Increasingly, many nutritional products are employing partially hy-... 

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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