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Posted on Thu, Jan 13, 2022

Purification Systems

Chromatography systems, or purification systems can be used to purify protein, nucleic acids, or just peptides. It comes in different sizes for different scales of purification. Research labs often do purification in smaller batches and in industry settings companies do large scale purifications. The AKTA pure is an example of one such useful technology for automating the purification process, avoiding human errors, keeping the purification at a regulated temperature such as  if you put the machine in a colder environment for less stable molecules, and having a consistent and regulated amount of pressure applied to purification columns.

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

Purification system components

The chromatography column is a column that is pre-packed with a specific type of resin. The type of resin depends on your purification needs. For example, a common resin is a nickel resin which binds to his-tags that are often placed on protein expression plasmids. The column is placed within the purification machine. It is important to understand how the machine works so we will go over the general structure of the machine. It begins with inlets where you can place buffers. You will use washing buffers to wash the column during the purification or an elution buffer to elute the protein from the column after it is bound and washed.

There is an injection valve where you inject your sample you wish to purify. From the injection valve a sample is injected into the purification column as a steady pressure to allow the sample time to bind to the Nickel in the column. Then the system pumps can inject more wash buffer into the column to wash away everything except the protein of interest, especially any non-specifically bound material. After the column the machine has a UV monitor that can detect the presence of proteins or other molecules. The ability of the UV monitor to absorbance depends on which wavelength of light the molecules in solution absorb. The last part of the machine is the fraction collector. The collector is programmed to collect solutions coming out of the column at regular intervals/volumes. The larger machine can do this process in parallel with parallelized inlet values and columns. 

Magnetic Bead alternatives

AKTA pure is one version of a chromatography for purification system that is a useful machine for purification for protein (described above) as well as nucleic acids and peptides. Laboratories and scientific companies also have the option to use magnetic beads for purification methods. Magnetic beads can be pre-conjugated to nickel-NTA (Ni-NTA) the same way that purification column resins are pre-packed with Ni-NTA.

The magnetic beads can then be used with a magnetic rack to do purification. We will describe the protocol briefly. Magnetic beads are first allowed to bind with the solution containing your protein or molecule of interest. The beads are then placed on a magnetic rack and stay there while the solution is exchanged with a wash buffer for washing steps. Lastly, an elution buffer is added to elute proteins from magnetic beads.

We have several articles you can read to learn more about magnetic bead purification and other purification techniques and pertinent information: "protein isolation protocol" or "protein expression and purification" or "protein purification system". Either column (chromatography) techniques and magnetic bead techniques have their advantages depending on what your lab needs and has access to. It is always good to know your options!

free protein purification handbook


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