AAT Bioquest

What is protein purification?

Posted August 12, 2020


Protein purification involves a sequence of steps to isolate one or more proteins from cells, tissues or whole organisms. Protein purification is important for the characterization of the function, structure and interactions of protein of interest. The purification process typically involves the separation of proteins from the non-protein mixture and subsequent separation of the protein of interest from other proteins in the mixture. This is one of the most challenging steps in protein purification.

Protein purification involves three major steps, these include:

  1. Extraction – If the protein of interest is not released by the cell into the surrounding culture medium, disruption of the cell membrane may be required to release the protein. This can be achieved using one of the following methods: sonication, homogenization, permeabilization by detergents or repeated freeze-thaw cycles.
  2. Precipitation and Differential Solubilization – To isolate proteins, precipitate by adding excess amounts of ammonium sulfate. Then collect the different fractions of precipitated protein and perform dialysis to remove ammonium sulfate.
  3. Chromatographic techniques – One of the most commonly used methods for purifying proteins. Proteins can be purified based on characteristics such as size, affinity and charge.

Additionally, other biological parameters such as enzymatic activity and immunoreactivity, can be used to assess the purity of an isolated protein of interest


Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 4.1, The Purification of Proteins Is an Essential First Step in Understanding Their Function. Available from: https://www.ncbi.nlm.nih.gov/books/NBK22410/

Scopes R.K. (1994) The Protein Purification Laboratory. In: Protein Purification. Springer Advanced Texts in Chemistry. Springer, New York, NY. https://doi.org/10.1007/978-1-4757-2333-5_1