Healthcare Technology Featured Article

July 30, 2019

An Overview of the Protein Purification Process




What does it mean to purify proteins? Protein purification is a necessary process to identify the structure and function of a protein prior to study. Protein purification involves several steps, from disrupting the cell and isolating the protein to removing impurities. You may need to undergo the protein purification process if you encounter samples with impurities or components you do not need. Take the following steps to successfully purify your protein.

The Importance of Protein Purification

It is not possible to characterize the interactions or functions of a protein without protein purification. This is because the protein could contain binding proteins or other components that alter its state. If you skip protein purification, then there is no way to accurately gauge the quality of the protein. You may then waste precious time and resources conducting your experiment on an impure protein, without realizing it until many steps down the line. Starting from purified proteins is the only way to accurately investigate its biochemical function and protein interactions.

Pick Your Process

There is more than one effective method for purifying a protein. Researchers today use several methods – most of which require high-quality protein extractions. Once you have carefully and successfully disrupted, isolated, and extracted your protein, use one of the following methods for purification:

  • Affinity chromatography. This is a single-step purification process that leads to a purity level that is typically good enough for analytical purposes. It uses molecular conformation to purify the protein.
  • Immunoprecipitation. Immunoprecipitation is usually a preemptive step before cell blotting or other purification techniques. It solidifies an antigen from immobilized protein antibodies using a solid support. Modern strategies use magnet beads.
  • Proteomics. Proteomics studies look at the total set of proteins in the organism’s body, as well as each protein’s function, structure, and numbers. This helps the researcher find the proteins of interest without wasted time.
  • Enzyme assay. An enzyme assay, or test, checks for a unique identifying property of the protein, so the researcher can detect when the protein is present. Most enzyme assays use the reaction the enzyme causes in the cell.

Purification often separates the non-protein parts from the protein sample. Then, it will serve to separate the protein you wish to study from other proteins. This final separation is typically the most time-consuming part of the purification process. The purification of proteins for analytical purposes usually results in a small sample for studying.

Determine the Success of Protein Purification

Once you conduct your protein purification, you should check to make sure the process worked. Monitor your protocol at each step using an SDS-PAGE analysis. This is a common technique that gives the researcher data about the protein’s size and quantity. Your analysis can measure parameters such as protein homogenization, ion-exchange chromatography, salt fractionation, and affinity. You should look at the total protein, total enzyme activity, specific activity, yield, and finally the purification level.

Calculating a protein’s purification level takes dividing the specific activity of the protein (found by dividing total activity by total protein) by the specific activity of the initial extract. The number of bands on your SDS-PAGE analysis should decrease correspondingly with the level of purification. The best purification processes end with both high levels of purification and reasonable yield. Using the right technologies can help you successfully purify proteins without wasting too much of your sample.

 

SOURCES:

http://www.emdmillipore.com/CA/en/life-science-research/protein-sample-preparation/protein-purification/Gdib.qB.6CIAAAE_ztx3.Mmz,nav 
https://www.ncbi.nlm.nih.gov/books/NBK22410/ 
https://www.thermofisher.com/us/en/home/references/protein-analysis-guide/affinity-chromatography/what-is-protein-purification.html 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076634/
http://www.bio-rad.com/en-us/applications-technologies/sds-page-analysis?ID=LW7FGX4VY








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