Protein Purification - Precipitation and Differential Solubilization

Precipitation and Differential Solubilization

In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH₄)₂SO₄. This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitate protein. Ammonium sulphate can be removed by dialysis.The hydrophobic groups on the proteins gets exposed to the atmosphere and it attracts other protein hydrophobic groups and gets aggregated. Protein precipitated will be large enough to be visible. One advantage of this method is that it can be performed inexpensively with very large volumes.

The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a particular membrane fraction can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein's native conformation during complete purification.