Methods for Protein Purification

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An important part of biotechnology research is to use protein engineering techniques to design or modify proteins with optimized properties for specific industrial applications. To do this scientist must be able to isolate and purify proteins of interest so their conformations, substrate specificities, reactions with other ligands, and specific activities can be studied.

The degree of protein purity required depends on the intended end use of the protein.

For some applications, a crude extract is sufficient. However, for other uses, such as in foods and pharmaceuticals, a high level of purity is required. To achieve this several protein purification methods are typically used, in a series of purification steps.

Each protein purification step usually results in some degree of product loss. Therefore, an ideal protein purification strategy is one in which the highest level of purification is reached in the fewest steps. The selection of which steps to use is dependent on the size, charge, solubility and other properties of the target protein. The following techniques are most appropriate for purifying a single cytosolic protein. Purification of cytosolic protein complexes is more complicated and usually requires that different methods be applied.

First Steps for Protein Purification

The first step in purifying intracellular (inside the cell) proteins is the preparation of a ​crude extract.

The extract will contain a complex mixture of all the proteins from the cell cytoplasm, and some additional macromolecules, cofactors, and nutrients. Crude extract may be used for some applications in biotechnology, however, if purity is an issue, subsequent purification steps must be followed. Crude protein extracts are prepared by removal of cellular debris generated by cell lysis, which is achieved using chemicals and enzymes, sonication or a French Press.

The debris is removed by centrifugation, and the supernatant is recovered. Crude preparations of extracellular proteins may be obtained by simply removing the cells by centrifugation.

For certain biotechnology applications, there is a demand for thermostable enzymes: Enzymes that can tolerate high temperatures without denaturing, and while maintaining a high specific activity. Organisms that produce them are sometimes called extremophiles. An easy approach to purifying a heat-resistant protein is to denature the other proteins in the mixture by heating, then cooling the solution (thus allowing the thermostable enzyme to reform or redissolve, if necessary. The denatured proteins can then be removed by centrifugation.

Intermediate Purification Steps

In the past, a common second step to purifying a protein from a crude extract was by precipitation in a solution with high osmotic strength (i.e. salt solutions). Nucleic acids in the crude extract can be removed by precipitating aggregates formed with streptomycin sulfate or protamine sulfate. Protein precipitation is usually done using ammonium sulfate as the salt.

Different proteins will precipitate in different concentrations of ammonium sulfate.

In general, proteins of higher molecular weight precipitate in lower concentrations of ammonium sulfate. Salt precipitation does not usually lead to a highly purified protein but can assist in eliminating some unwanted proteins in a mixture and concentrating the sample. Salts in the solution are then removed by dialysis through porous cellulose tubing, filtration, or gel exclusion chromatography.

Modern biotech protocols often take advantage of the many commercially-available kits that provide ready-made solutions for standard procedures. Protein purification is often performed using filters and prepared gel filtration columns. All you have to do is follow the instructions and add the right volume of the right solution and wait the specified length of time while collecting the eluant (what comes out the other end of the column) in a fresh test tube.

  • Chromatographic methods can be applied using bench-top columns or automated HPLC equipment. Separation by HPLC can be done by reverse-phase, ion-exchange or size-exclusion methods, and samples detected by diode array or laser technology. 

    Protein Visualization and Assessment of Purification

    • 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 the 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 collected 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 gives the purest results and highest specific activity compared to other techniques.
  • SDS-PAGE is polyacrylamide gel electrophoresis, performed in the presence of SDS (sodium dodecyl sulfate) which binds to proteins giving them a large net negative charge. Since the charges of all proteins are fairly equal, this method separates them almost entirely based on size. SDS-PAGE is often used to test the purity of protein after each step in a series. As unwanted proteins are gradually removed from the mixture, the number of bands visualized on the SDS-PAGE gel is reduced, until there is only one band representing the desired protein.
  • Immunoblotting is a protein visualization technique applied in combination with affinity chromatography. Antibodies for a specific protein are used as ligands on an affinity chromatography column. The target protein is retained on the column, then removed by rinsing the column with a salt solution or other agents. Antibodies linked to radioactive or dye labels aid in the detection of the target protein once it is separated from the rest of the mixture.


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.