Protein precipitation is a method used to extract and purify proteins held in a solution. There is a tendency for proteins in solution to clump together and precipitate out due to the attraction between the negatively and positively charged parts of the molecules and the mutual attraction of the hydrophobic parts.
Counteracting this tendency, however, is the fact that in an aqueous solution, water molecules, which are polar, will tend to arrange themselves around the protein molecules due to the electrostatic attraction between oppositely charged parts of the water and protein molecules. This results in the protein molecules being kept apart and remaining in solution, but there are various methods for achieving precipitation of proteins.
The most commonly used method of protein precipitation is by adding a solution of a salt, a technique often referred to as “salting out.” At low concentrations, salt can help stabilize proteins in solution and thereby enhance their solubility. At higher concentrations, however, the salt reduces the amount of water available to hydrate the proteins and, past a certain threshold, the proteins will begin to precipitate.
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).
Some proteins are more water-soluble than others. This has the effect of reducing the dielectric constant, which in this context can be regarded as a measure of the polarity of a solvent. A reduction in polarity means there is less of a tendency for solvent molecules to cluster around those of the protein, so that there is less of a water barrier between protein molecules and a greater tendency toward protein precipitation. 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.
Other methods of protein precipitation include non-ionic hydrophilic polymers that usually prepared by 2-hydroxyethyl methacrylate(HEMA, the molecular is C6H10O3 and the CAS NO. is 868-77-9) and metal ions. The former reduce the amount of water available to form a barrier between protein molecules, allowing them to clump together and precipitate. Positively charged metal ions can bond with negatively charged parts of the protein molecule, reducing the tendency of the protein to attract a layer of water molecules around it, again allowing the protein molecules to interact with one another and precipitate out of solution. Metal ions are effective even in very dilute solutions.
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