It is well known that most recombinant protein purifications are mainly done through different types of chromatography, explained in our protein purification handbook. However, the use of magnetic particles is a very interesting alternative to these techniques, providing great advantages and simplifying the process in many aspects. The necessary equipment for purification with magnetic particles is simple: we need a magnet or any device capable of creating a magnetic field, and the particles themselves.
Each magnetic particle contains a nucleus with magnetic properties, which constitutes the base of the structure. The nucleus is generally formed by magnetite (Fe3O4), which has superparamagnetic properties, or by maghemite (Fe2O3), with ferromagnetic properties.
This post is an excerpt from our protein purification handbook, which explains the basics of recombinant protein purification. Download The Basic Guide to Recombinant Protein Purification here:
But...what is the difference between superparamagnetism and ferromagnetism?
On the one hand, superparamagnetism is the phenomenon that occurs when the dipole moment of a single-domain particle rapidly fluctuates in its nucleus. This fluctuation is due to the thermal excitement, so there is no magnetic moment in a temporary microscopic scale. In this way, particles with superparamagnetism are not magnetic when an external field is applied to them, but they develop a magnetic moment through this situation.
The advantages of superparamagnetic particles are their easy resuspension, large surface area, slow sedimentation and even distribution in the medium. When they become magnetized, they behave as small permanent magnets, forming aggregates or reticles due to the magnetic interactions.
On the other hand, ferromagnetism confers the particles a permanent magnetic moment, which exceeds the fluctuation caused by the thermal excitement. This means that the ferromagnetic particles have strong magnetic properties, allowing an easy separation when an external field is applied, even in a viscous medium.
As part of the coating, magnetic particles usually have a layer of an inorganic polymer, apart from the radical groups. This inorganic polymer is included in order to eliminate hydrophobicity and avoid unintended aggregation.
Aside from having a magnetic nucleus and the polymeric layer, magnetic particles have a coating that has to be designed to specifically bind the protein to be purified. There are magnetic particles designed to capture proteins with histidine tags, and they are covered by cobalt or nickel ions for this purpose. By adding the particles in the medium, the histidine tag of our protein will interact with the ions on the particles, allowing for an easy, fast capture and separation.
Other particles have chemical radicals on their surface to decorate them as needed (for example, a specific antibody to our target protein can be coated onto the particle surface).
As in the case of chromatography, that has many variants and different techniques, there are many types of magnetic particles that can be personalized and decorated as needed for each experiment and application.
How are these magnetic particles obtained?
Dedicated companies sell magnetic particles already functionalized (they have a specific name for each one), but they also sell blank particles without functional groups (for example, a coated particle with –COOH or –NH2 group). These last ones can be coated by using such functional groups through easy chemical reactions. These groups allow ligand binding and decorate the particle. Examples of them are antibodies, enzyme substrates, streptavidin, G or A proteins (that specifically bind IgG) or amylose (for proteins that are Maltose Binding Protein, MBP).
Therefore, another great advantage of the magnetic particles is their great versatility derived from the possibility to personalize them.
Researching in order to find the optimal design of the magnetic particle allows carrying out the separation in optimal conditions. The inclusion of specific antibodies or selective ions for histidine residues provides an increased level of purity in a relatively simple procedure in comparison to conventional chromatography. However, we need to remember that due to its low binding capacity to the ligand, magnetic particles are focused on the purification of small amounts of proteins, reserving the uses at an industrial scale for the chromatography techniques.
Did you find this post interesting? You can check this related post in order to learn more about protein purification:
- An insight into our protein purification handbook
- Polishing: the secret to achieve maximum protein purification
- How to purify recombinant proteins through magnetic particles