Salting out of proteins using ammonium sulfate precipitation

Methods Enzymol. 2014;541:85-94. doi: 10.1016/B978-0-12-420119-4.00007-0.


Protein solubility is affected by ions. At low ion concentrations (<0.5 M), protein solubility increases along with ionic strength. Ions in the solution shield protein molecules from the charge of other protein molecules in what is known as 'salting-in'. At a very high ionic strength, protein solubility decreases as ionic strength increases in the process known as 'salting-out'. Thus, salting out can be used to separate proteins based on their solubility in the presence of a high concentration of salt. In this protocol, ammonium sulfate will be added incrementally to an E. coli cell lysate to isolate a recombinantly over-expressed protein of 20 kDa containing no cysteine residues or tags.

Keywords: Ammonium sulfate; Cell lysate; Dialysis; Hofmeister series; Protein solubility; Salting-out.

MeSH terms

  • Ammonium Sulfate / chemistry*
  • Chemical Precipitation*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / isolation & purification
  • Osmolar Concentration
  • Proteins / chemistry*
  • Proteins / isolation & purification*
  • Solubility


  • Escherichia coli Proteins
  • Proteins
  • Ammonium Sulfate