by Luca Montore
Fun Rating: 4/5
Difficulty Rating: 3/5
What is the general purpose?
Protein purification is a technique that allows scientists to isolate a protein of interest from the complex mixture of molecules within cells.
Why do we use it?
Protein purification involves many steps, and when successful, it can allow for incredible leaps in scientific knowledge. Protein purification allows for the isolation of a protein, giving scientists the opportunity to do a wide variety of experiments and learn more about a specific protein without other interfering cellular components. These experiments can help scientists determine a protein’s role in the cell and determine how it functions. These experiments can also help identify the structure of a protein and other important characteristics that can not normally be determined when a protein is not isolated from all other components that are within a cell.
How does it work?
Protein purification involves 7 major steps:
The 7 steps of protein purification.
Created by author in BioRender.com
1 – Transformation.
First, a scientist needs to create a plasmid. A plasmid is a circular piece of DNA. This plasmid must include their gene of interest. A gene is a subsection of DNA that holds the information required for a cell to make a specific protein. Think of DNA like a massive cookbook and a gene is one recipe within the cookbook that produces a dish, or in this case a protein. There are many ways to create a plasmid that includes a specific gene of interest, but a common way is to utilize another technique called gene cloning or to buy the plasmid online. Once the plasmid has been created the next step is to insert the plasmid into a bacterial cell. This is called transformation.
2 – Bacterial Selection.
Once a plasmid has been inserted into bacteria, scientists place the bacteria onto an agar plate. These agar plates are filled with antibiotics that ensure only bacteria carrying the plasmid can grow. This is due to the plasmid carrying not only the gene of interest but a gene containing the information for destroying the antibiotic in the agar.
3 – Protein Production.
Once bacteria with the plasmid has grown on the agar plate, the next step is to transfer the bacteria to a larger flask containing nutrients for the bacteria to grow. This allows the bacteria to multiply and produce the protein of interest.
4 – Cell Lysis
The next step is to add a lysis buffer to the cells. A lysis buffer is a solution that breaks open the cellular membrane of the bacterial cells, allowing all of the contents, including the protein of interest to float around freely in the cell lysis solution.
5 – Load Cell Lysis
Once the bacterial cells have been lysed, they are added to a protein buffer. The protein buffer is a solution that helps stabilize the protein and prevents it from breaking down when it is outside of a cell. Proteins do not like to be outside of cells, so generating a protein buffer that allows the protein of interest to be stable is very important.
Once the cell lysis is in the protein buffer it is added to a column containing resin. Resin is a material used to separate proteins from other cellular components. The protein is separated from all other cellular components because the protein binds preferentially to the resin while all other molecules will flow through the column.
6 – Wash Column
Next, more protein buffer is added to the column to ensure no other cell debris, proteins, or other bacterial cell components have bound to the resin.
7 – Elute Protein
Finally, an elution buffer is added to the column. This buffer breaks the interaction between the protein and the resin. This allows the protein to fall off the resin and flow through the column.
The protein has now been purified!
