What Are The Techniques Involved In Protein Purification?

protein purification methods

The intended aim of protein purification is to remove degraded products and impurities. People also refer to protein purification as protein fractionation. However, what differentiates a protein purification lab from the usual lab is purification methods. There are different protein purification techniques, and we’ll discuss them in this article. 

What is Protein Purification? 

A protein is a polymer of amino acids organized in polypeptide chains. They are essential for the structure and function of cells. Before studying them, it is necessary to identify and isolate proteins. So, biochemists use a technique called protein purification. 
 Protein purification is a set of steps for separating one or a few proteins from a complex mixture. These mixtures comprise cells, tissues, or whole organisms. Purification is essential to figure out its function, structure, and interactions. 

Moreover, this process can separate the mixture into protein and non-protein parts. Usually, the most time-consuming part of protein purification is separating one protein from the rest. There are several reliable protein expression and purification services one could trust online if there are protein purification needs. But do thorough research before choosing a service.

Techniques Involved In Protein Purification

There are four techniques for protein purification. Read on as we have discussed them below.


You must get the material from which you intend to isolate the substance before purifying the protein. Historically, researched protein depends on its quantity and ease of separation. There are various ways to do this, including repeated freezing and thawing.

Also, the method of choice depends on the fragility of the protein and the strength of the cells.

After the extraction procedure, the solvent will include soluble protein. We can centrifuge the protein to separate it from cell membranes. Proteases are also extracted, and they begin digesting the proteins in the solution.

Further, if the protein is sensitive to proteolysis, it’s usually best to move fast and ensure the extract stays cold, thus, preventing it from breaking down.


Chromatography is a common way to get the protein sample you want. It puts compounds into groups based on how different they are. Also, it is a famous purification method that uses resin-filled columns to remove the protein you want from them.

Chromatography has two parts: a stationary phase and a moving phase. The stationary phase is the resin in the column, and the moving phase is the liquid solvent that goes through the column.

The protein of interest binds to the ligand in the stationary phase, while each mobile phase eliminates impurities. However, several chromatographic methods are used to separate proteins from other substances. Some of them are:

  • Ion Exchange Chromatography: This method differentiates different proteins based on their net charge.
  • Hydrophobicity: There are two methods in this chromatographic technique. They are hydrophobic interaction chromatography and reverse-phase chromatography.
  • Gel filtration: This approach separates bigger proteins from smaller proteins using a small crude extract. Because the solutes do not interact with the stationary phase, they have high sensitivity and do not cause sample loss.
  • Affinity Chromatography: Affinity chromatography is the most selective chromatography technology, and it produces the purest results; thus, they use it to complete the protein purification process.


Salt precipitation is another way to purify the protein. However, it is no longer popular because it doesn’t always give very pure proteins. Precipitation is possible by gradually adding more ammonium sulfate to the solution. Then, it collects the different parts of the precipitate protein. This approach has the advantage of handling massive volumes of data at a low cost.

They start with water-soluble proteins then move on to other types of proteins. If you want to separate integral membrane proteins, you’ll have to break the cell membrane. Sometimes, it’s possible to separate specific membrane traction before purifying a protein. Different proteins precipitate at different ammonium sulfate concentrations. The residual ions in the supernatant are removed by dialysis and filtration after the protein has been poured.


Centrifugation is the process of separating mixtures of particles that have different weights. It happens when a container with many proteins is rotated at high speeds. Then, the angular momentum of each particle causes an outward force that is proportional to its weight.

The resistance that the liquid puts on the particle offsets the inclination to travel through the liquid due to this force. Big particles move outward quicker than small particles in the liquid when the sample is “spun” in a centrifuge.

When particles are “spinning” in a centrifuge, a “pellet” may form at the bottom of the tank. This “pellet” comprises the most significant and least-draggable particles. These particles are the “supernatant,” containing non-compacted particles that stay in liquid. You can take the supernatant out of the vessel to separate it from the pellet.


Protein purification is now an essential part of biomedical research. With more protein discoveries, it’s necessary to look at how they look, how they work, and how they interact with other biomolecules.