- Reverse-phase chromatography (RPC) separates proteins based on their relative hydrophobicities. This technique is highly selective but requires the use of organic solvents. Some proteins are permanently denatured by solvents and will lose functionality during RPC, therefore this method is not recommended for all applications, particularly if it is necessary for the target protein to retain activity.
- Ion-exchange chromatography refers to separation of proteins based on charge. Columns can either be prepared for anion exchange or cation exchange. Anion exchange columns contain a stationary phase with a positive charge that attracts negatively charged proteins. Cation exchange columns are the reverse, negatively charged beads which attract positively charged proteins. Elution of the target protein(s) is done by changing the pH in the column, which results in a change or neutralization of the charged functional groups of each protein.
- Size-exclusion chromatography (gel filtration) separates larger proteins from small ones, since the larger molecules travel faster through the cross-linked polymer in the chromatography column. The large proteins do not fit into the pores of the polymer whereas smaller proteins do, and take longer to travel through the chromatography column, via their less direct route. Eluate is collected in a series of tubes separating proteins based on elution time. Gel filtration is a useful tool for concentrating a protein sample, since the target protein is collect in a smaller elution volume than was initially added to the column. Similar filtration techniques might be used during large scale protein production because of their cost-effectiveness.
- Affinity chromatography is a very useful technique for "polishing" or completing the protein purification process. Beads in the chromatography column are cross-linked to ligands that bind specifically to the target protein. The protein is then removed from the column by rinsing with a solution containing free ligands. This method generally gives the purest results and highest specific activity compared to other techniques.
Protein Visualization and Assessment of Purification
Zubay G. 1988. Biochemistry, 2nd Edition. Macmillan Publishing Co., New York, NY, USA.
Amersham Pharmacia Biotech. 1999. Protein Purification Handbook, Edition AB. Amersham Pharmacia Biotech Inc. New Jersey, USA. http://www.biochem.uiowa.edu/donelson/Database%20items/protein_purification_handbook.pdf.