Fractional precipitation is frequently used at laboratory scale to remove gross impurities from small sample volumes and occasionally used in small-scale commercial production. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure 75.
Figure 75.Three ways to use precipitation.
Details taken from:
Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998).
Some proteins may be damaged by ammonium sulphate. Take care when adding crystalline ammonium sulphate: high local concentrations may cause contamination of the precipitate with unwanted proteins.
For routine, reproducible puriﬁcation, precipitation with ammonium sulphate should be avoided in favor of chromatography.
In general, precipitation is rarely effective for protein concentrations below 1 mg/mL.
Solutions needed for precipitation:
*The % saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.
The quantity of ammonium sulphate required to reach a given degree of saturation varies according to temperature. Table 14 shows the quantities required at +20 °C.
Table 14.Quantities of ammonium sulphate required to reach given degrees of saturation at +20 °C.