Most current protein purification methods use agarose beads carrying affinity functionalities such as IMAC, Glutathione, or antibodies. The choice of these functional groups depends on the protein of interest to be purified, and a large variety is available, including pre-functionalized beads that can be coupled to biomolecules (see SEPMAG® protein purification handbook chapter 4 and 5).
Magnetic beads vs Agarose beads: Use of Agarose beads
Agarose is most frequently used as protein purification matrix for two reasons. First, agarose shows very low non-specific binding to proteins, increasing the purity of the eluted protein fraction. Secondly, agarose particles as a whole consist of a hydrophilic, three-dimensional mesh large enough for proteins to diffuse into. This mesh allows biomolecules to interact with the first micrometers of the outer shell of the agarose particle, providing a large interaction surface between the functional groups and the proteins of interest, thereby increasing the yields that can be obtained in protein purification.
You will find this post and many more interesting articles about using biomagnetic separation for protein purification in our Advanced Guide to Biomagnetic Protein Purification:
Agarose matrices are available both non-magnetic and with a magnetite (Fe3O4) core that renders them magnetic. Non-magnetic agarose beads are typically larger (40-150 µm in diameter) than magnetic beads to reduce back-pressure in chromatography. Magnetic beads are typically smaller (2-40 µm in diameter). Smaller beads provide more surface area and therefore higher interaction with the protein of interest. However, for best magnetic behavior a minimal size of 10 µm should be used, especially when working with ferrimagnetic beads (see SEPMAG® protein purification handbook chapter 12).
For maximum flexibility, the chemistry of the affinity functions, as well as the surface, should be identical between magnetic and non-magnetic materials, allowing for easy switching between the two systems.
Difference between non-magnetic beads vs agarose beads
The main difference between non-magnetic beads vs agarose beads is the way they are separated from the surrounding medium. In the first purification steps, this medium can be bacterial or cell lysate, cell culture medium or any kind of physiological buffer. During the wash and elution steps of protein purification, this medium contains buffers of different properties (e.g. salt concentrations, pH) that induce binding of the specific protein, removal of contaminants, and elution of the protein of interest. Viscosity of these solutions can vary, e.g. when substances like glycerol are added to reduce non-specific binding. Every protein purification process requires several separation steps, therefore efficient separation has a large impact both on processing time, and purification success.
For all protein purification experiments, it is important to adjust the amount of affinity beads to the amount of protein in the starting material. This is important not only for cost reasons: too little affinity material will result in incomplete binding of the protein in the solution. Too many affinity binding sites offered, on the other hand, will lead to binding of other proteins, making the purification less specific, so that additional purification steps are required in order to obtain pure protein.
Non-magnetic agarose beads can be separated from the medium by centrifugation, by filtration in gravity columns, or on chromatography systems. Switching between these methods (e.g. when moving to higher purification scales) requires optimization. When protein expression levels are low, large amounts of starting material need to be applied to rather small volumes of agarose beads, which can be a time-consuming process. For very sensitive applications requiring only microliters of volumes, such as immunoprecipitation, separation could be incomplete, leading to either loss of material or contamination with agarose beads.
IDA vs. NTA. Chelating ligands nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA) support similar interaction between Ni2+ and imidazole rings of a polyhistidine tag, but NTA coordinates the Ni2+ with 4 valencies and IDA with only 3 (orange circles). This difference impacts the quality of the resulting purified protein fraction.
With magnetic beads, the principle of separation stays the same regardless of application and volumes. Test reactions or sensitive applications like immunoprecipitation can be performed in few microliters, and even automated on microliter robots. Purification of proteins from liters of medium or cell lysate can be done equally well because the handling volumes are reduced significantly after the first separation step.
A simple protocol for using magnetic beads
- Mix solution containing molecules of interest with magnetic beads. Allow time for binding to occur.
- Place your container of solution on a magnetic rack. Remove solution from the container.
- Add a wash buffer to clean the beads bound to your molecules of interest.
- Repeat wash 2 to 3 times.
- Add an elution buffer to elute molecules from beads. Remove solution from container.
There are many types of pre-conjugated beads. Beads are pre-conjugated to molecules that will bind your molecules of interest. Let’s go over some examples. If you are interested in binding antibodies, you can purchase magnetic beads pre-conjugated to protein A, protein G, or a hybrid protein A/G. If you are interested in binding a his-tagged protein, you can order Nickel conjugated magnetic beads. Silicon coating can make beads that bind to DNA or RNA. Now that there are many sizes of magnetic separation systems available, you can adjust your protocol to purify or isolate large amounts of protein or small amounts of RNA, whatever your need.
Read more at https://cube-biotech.com/products/magnetic-beads/
Author: Dr. Roland Fabis, Head of Operations at Cube Biotech GmbH, Monheim, Germany
Dr. Roland Fabis holds a PhD in inorganic chemistry from the University of Muenster, Germany, where he worked on silicon-organic compounds, surface modification with silanes, and applications of these substances. Dr. Fabis joined the QIAGEN headquarters in Hilden, Germany in 1995, where he held various positions in basic research, product development and technology transfer, especially in the fields of magnetic particle synthesis and protein purification. He is one of the co-founders of Cube Biotech and responsible for the development and production of protein affinity materials, including manufacturing of magnetic beads.