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Posted on Thu, Apr 01, 2021

Protein purification system

Proteins are one of the four main macromolecules in biology. They have many crucial functions in all living organisms. Research laboratories all over the world study the many types of proteins to better understand the mechanisms of living things

In order to study proteins, their interacting partners, and their functions, it is necessary to purify them. For this, it is important to develop a protein purification system and protocol. Before protein purification, the first step will always be to lyse cells that are expressing your protein. You can read more about lysing cells in this article: Cell Lysis Buffer. A major protein purification system is called chromatography, where a protein is selected for inside of a column then slowly eluted out in aliquots. The main types of chromatography is affinity chromatography, ion exchange, and size exclusion chromatography. 

Free PDF guide:  "Basic Guide to Recombinant Protein Purification" 

Examples of protein purification system

An example of how an affinity column works is the interaction of Ni-NTA (nickel-Nitrilotriacetic acid) and HIs-Tags (6-histidine tag). Many protein expression systems will be a plasmid with a 6-histidine tag at the end of the protein sequence. His-tags bind strongly to Ni-NTA in the presence of a good binding buffer. For the protein purification system, the columns need to be cleaned between use with buffers and equilibrated with your buffer of choice for purification. Then the lysis solution containing your protein runs through the column. Wash steps are important to wash away non-specific binding on the column. The amount of washes and length of the washes vary depending on the column. Lastly, an elution buffer will be used to elute your protein from the column.

Magnetic beads as a protein purification system

Laboratoratories now also use magnetic beads as protein purification systems. Magnetic beads can be used to create the same interactions as columns, such as being pre-conjugated with Ni-NTA, but have much simpler protocols. Proteins are often purified from large quantities of cell lysis which is now possible to do with modern separators accommodating up to 10 liters of solution to separate. These separators even allow you to monitor the separation process, giving you control over the purification process. 

Final thoughts for choosing a protein purification system

There are several factors that will impact your choice of protein purification system. Your lab might have a system in place already. Researchers or labs that historically always used affinity chromatography columns, will continue with those columns because they have the infrastructure to do it that way. The technology for magnetic bead purification has made possible a protein purification system that is efficient and gives good yield. When creating new protocols for new protein purifications, many labs now look to magnetic bead protein purification systems. 

 

free protein purification handbook

 

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