Methods for Protein Purification in Biotechnology

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An important component of biotechnology research is the use of protein engineering techniques to design or modify proteins. These protein purification techniques optimize protein properties for specific industrial applications.

These techniques require scientists to isolate and purify proteins of interest so that their conformations and substrate specificities can be studied. Also requiring study are the reactions with other ligands (a protein that attaches to a receptor protein) and specific enzyme activities.

The degree of protein purity required depends on the intended end use of the protein. For some applications, a crude extract is sufficient. Other uses, such as in foods and pharmaceuticals, a high level of purity is required. Several techniques for protein purification are used to reach a required purity level.

Develop a Strategy

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.​

Prepare a Crude Extract

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.

This 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 the removal of cellular debris generated by cell lysis, which is achieved using chemicals, enzymes, sonication or a French Press.

Remove Debris From the Extract

The debris is removed by centrifugation, and the supernatant (the liquid above a solid residue) is recovered. Crude preparations of extracellular (outside the cell) 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, while maintaining high specific activity.

Organisms that produce heat-resistant proteins 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 Protein Purification Steps

Modern biotech protocols often take advantage of the many commercially available kits or methods that provide ready-made solutions for standard procedures. Protein purification is often performed using filters and prepared gel-filtration columns.

Follow the dialysis kit's instructions and add the right volume of the right solution and wait for the specified length of time while collecting the eluant (the solvent passed through the column) in a fresh test tube.

Use Chromatographic Methods

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. ​

Employ Precipitation

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). Protein precipitation is usually done using ammonium sulfate as the salt. Nucleic acids in the crude extract can be removed by precipitating aggregates formed with streptomycin sulfate or protamine sulfate.

Salt precipitation does not usually lead to a highly purified protein but can assist in eliminating some unwanted proteins in a mixture, and by concentrating the sample. Salts in the solution are then removed by dialysis through porous cellulose tubing, filtration, or gel exclusion chromatography.

Different proteins will precipitate in different concentrations of ammonium sulfate. In general, proteins of higher molecular weight precipitate in lower concentrations of ammonium sulfate.

Protein Visualization and Assessment of Purification

Reverse-phase chromatography (RPC) separates proteins based on their relative hydrophobicities (exclusion of non-polar molecules from water). 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 and Gel Filtration

Cation exchange columns are the reverse, negatively charged beads that attract positively charged proteins. Elution (extracting one material from another) 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

Size-exclusion chromatography (also known as gel filtration) separates larger proteins from smaller 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 a less direct route.

Elution Time

Eluate (the result of elution) 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 and Electrophoresis

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 the highest specific activity compared to other techniques.


SDS-PAGE (sodium dodecyl sulfate used with polyacrylamide gel electrophoresis) 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.

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Phillips, Theresa. "Methods for Protein Purification in Biotechnology." ThoughtCo, Aug. 9, 2021, Phillips, Theresa. (2021, August 9). Methods for Protein Purification in Biotechnology. Retrieved from Phillips, Theresa. "Methods for Protein Purification in Biotechnology." ThoughtCo. (accessed June 9, 2023).