Question

Explain how you would use salting out to prepare a more concentrated solution of a protein.

Short answer

Many undesirable proteins are removed from a solution containing a variety of proteins by lowering the salt concentration to just below the precipitation point of the protein to be purified. The salt concentration of the resulting solution is then raised to precipitate the target protein after the precipitated proteins have been removed by filtration or centrifugation. This process will purify and will give a concentrated solution of desired protein.

Step-by-step solution

Factors that affect proteins solubility

Because a protein has numerous charged groups, its solubility is affected by dissolved salt concentrations, solvent polarity, pH, and temperature. Some of these variables can be changed to cause specific proteins to precipitate while other proteins remain soluble.

Salting out

Salting out is a purification procedure that takes advantage of particular compounds' reduced solubility in a solution with a very high ionic strength. The salting out effect occurs due to competition for solvent molecules between the added salt ions and the other dissolved solutes.

Fig: Fractionation by salting out.

(a) Salt is added to solution of mixed protein molecules at a concentration which is slightly below the salting-out point of the target protein (Blue).

(b) Unwanted proteins (Red) are precipitated which are discarded. More salt is added in the supernatant to bring the concentration which is sufficient to precipitate the target protein.

Explanation(c) After centrifugation, the target protein is recovered as precipitate and added in suitable buffer.

Explanation

So many of the additional ions are solvated at very high salt concentrations that there is substantially less bulk solvent available to dissolve other molecules, including proteins. Salting out is the basis of one of the most widely used protein purification techniques because various proteins have distinct ionic and hydrophobic compositions and so they precipitate at varying salt concentrations.

So, if we increase the salt concentration of the solution to a point which is higher than the salting-out point for the desired protein, we can obtain a concentrated solution of a desired protein.