What Are the 4 Methods of Protein Purification?

Proteins are among the most numerous and essential molecules in the human body. In fact, researchers have identified at least 20,000 unique human proteins with as many as 42 million protein molecules in each human cell. The study of these proteins is a crucial element in many scientific fields – including drug discovery and biopharmaceutical development.

Four Methods of Protein Purification

How are researchers able to accurately determine the structure of such a small part of a cell and track its specific function within the cell? First, researchers must purify a sample of protein to isolate the desired type of protein that they are trying to examine. In general, researchers utilize one of four major methods of purification to isolate proteins for research. Protein purification methods include:

  1. Extraction. This method involves separating protein from the cells that contain it and placing it into a solution. Depending on how hardy or fragile the protein is, the method of extraction can vary but usually involves breaking or damaging the cells containing the protein to force them to release the protein molecules. Extraction methods include the use of organic solvents, sonication (agitation by sound waves), pressure homogenization, and repeated freezing/thawing.

    After the protein has separated from the cell via extraction, it is contained in a solution with other cell matter. In most cases, researchers place the solutions in a centrifuge to separate proteins from the other components using centrifugal force so that they do not become damaged.
  2. Precipitation and Differential Solubilization. Another common method for protein purification involves “salting out” the protein by adding ammonium sulphate. As researchers increase the amount of ammonium sulphate in a solution, the electronic charges on the surfaces of the protein interact with the salt. Different hydrophobic patches on the protein are exposed, and proteins become attracted to each other.

    As a result, proteins slowly aggregate by type and fall out of the solution, a process known as precipitation. Based on different levels of solubilization, researchers can isolate different types of proteins. This method can be used to purify large amounts of protein at a time.
  3. Ultracentrifugation. This method of protein purification involves the use of a centrifuge to spin tubes of solution at a rapid pace. The momentum of the centrifugal force acts proportionally upon each molecule in the solution, dependent on its mass. In addition, the liquid in the solution resists the movement of molecules proportionally, based on mass.

    When researchers spin protein samples in a centrifuge, more massive particles with low drag gather at the bottom. As the sample reaches equilibrium, the particles within are layered. The protein can then be collected and separated from other molecules.
  4. Chromatography. This involves forcing a protein solution through a column containing specially packed materials. As the proteins interact with the column materials, they can be identified and separated by the time they took to pass through the column.

    Multiple types of chromatography exist, and the type of chromatography that is chosen is dependent on which particular protein a researcher is trying to purify. For example, size exclusion chromatography can be used to isolate proteins based on the amount of time it takes them to emerge from a gel. In contrast, ion exchange chromatography isolates proteins based on the attraction between the column materials and the proteins. Finally, affinity chromatography uses ligands that bind with specific proteins to separate desired proteins from a solution. 

Depending on the type of protein desired, researchers may employ one or more of the above methods. The result is a purified protein sample that can be used  for more extensive research and tracking. Once protein purification has occurred, researchers can successfully work to identify the protein functions that exist in various diseases and uncover druggable solutions to combat their effects.