Guide to magnetic beads / MagBeads for protein purification

The workflow with MagBeads is surprisingly easy. To achieve protein purity, these 6 easy to perform steps have to be performed that are depicted in figure 1.
MagBeads workflow Fig. 1: Workflow of a standard protein purification procedure using magnetic beads/MagBeads. Detailed specifications of the individual steps on how to use MagBeads are provided in the following paragraph.

Workflow of MagBead protein purification step-by-step in a listed guide

  • Step 1:
    The usual starting point of a MagBead protein purification is a cell lysate in which the proteins of interest are mixed together with every other proteins the expression cells had produced simultaneously.
  • Step 2:
    Addition of an appropriate amount of MagBeads to the lysate. We suggest that you use sterile pipette tips for this.
  • Step 3:
    Shaking of the MagBead/Protein mixture. We suggest automated mixing with a slower but steady speed. No vortexing! A duration of around 2 hours at room temperature is what we see as sufficient. Figure 1 may only show a micro centrifuge tube but a sterile falcon tube is also as well suited.
  • Step 4:
    Now, almost every protein of interest should have bound to a MagBead. Again, it is important to use more MagBeads than protein of interest is present to ensure maximum binding effectiveness.
  • Step 5:
    Apply a magnetic force to your MagBeads to separate them and in turn the protein of interest from the rest of the lysate. For micro centrifuge tube we suggest our practical MagBead separator for this.
  • Step 6:
    In the final step the rest of the cell lysate that did not bind to the MagBeads will be removed using again sterile peptide tips. Keep the magnetic force active to avoid accidental removal of some MagBeads.
  • Optional:
    Additional washing steps will increase the purity of your protein. Too see what washing buffer in appropriate to what kind of MagBead check our protocols & datasheets page for the corresponding PDF-files.
    You will also find the necessary information to elute the proteins of interest from the MagBeads later on if this is necessary for you. Feel free to contact us in case you have any further questions.

The advantages of magnetic beads / MagBeads in detail

Using magnetic beads for protein purification has a number of advantages compared to non-magnetic affinity matrices. At the same time, there are a lot of different products available with different features and benefits. This short summary shall support you in finding the optimal product for your research.
 
Magnetic Beads; microsope; reality Fig. 3: PureCube MagBeads at 100x magnification.
GFP-ReaderFig. 2: Scheme of a magnetic bead. The magnetic cores are all very consistent.
 
 
 

How can magnetic beads purify proteins?

The magnetic bead itself cannot interact with a protein. It has to be loaded with ions. Different metal ions result in differing affinity and specificity for a his-tagged protein (See our overview). The most used tool to purify proteins via affinity chromatography is the nickel-NTA ligand (Fig. 2). The nitrilotriacetic acid (NTA) ligand is coupled to matrices like magnetic beads and then loaded with ions. The ions interact with specific tagged proteins (e.g. 6x his tagged proteins).In figure 4 nickel ions are loaded to a magnetic bead. Although the nickel ions are light blue the magnetic beads appear black because of the magnetic cores.
Ni-NTA Magentic Bead Scheme
Fig. 4: To purify proteins different ligands with various ions are coupled to the magnetic bead.

Magnetic Core

MagBeads used in protein purification contain a magnetic core made of magnetite which is covered in different materials. MagBeads are typically either ferri (or ferro-) magnetic, or superparamagnetic.
 
  • Ferri/Ferromagnetic magnetic cores are typically large (>30 nm) and show a strong magnetic moment. They retain this magnetic moment even after removal of the magnetic field. This effect is called "magnetic remanence". The strong magnetic field leads to a fast separation of the beads in the magnetic field. At the same time, they sometimes show self-magnetism and may attach to metal surfaces.
 
  • Superparamagnetic magnetic cores are smaller (5-30 nm), and their magnetic moment is weaker. When the outer magnetic field is removed, the beads lose their magnetism. Separation of the beads in the magnetic field typically takes longer or is less efficient. At the same time, use with metal surfaces is facilitated.
AGM measurement of magnetic beads
Fig. 5: Alternating gradient magnetometer (AGM) measurement of PureCube MagBeads.
X-axis: applied magnetic field.
Y-axis: Resulting magnetic moment of the dried MagBead particles
M. Remanence: Residual magnetism of the particles in absence of an external magnetic field.
Saturation: Maximal magnetic moment of the particles.
PureCube MagBeads are ferrimagnetic and have a strong saturation signal, but show a very low remanence in the absence of a magnetic field, preventing bead aggregation. Data kindly provided by Dipl. Ing. Moritz Ebeler, Karlsruhe Institute of Technology, Germany

Surface Chemistry

The magnetic core can be covered in a range of different materials, providing different properties.
 
  • Agarose forms a three-dimensional hydrophilic mesh with neutral charge. Similar to non-magnetic agarose, it is very well suited to bind to proteins, e.g. via affinity ligands. The large interacting surface leads to high binding capacities. The neutral surface reduces non-specific binding.
  • Polyvinyl beads have a carboxylate surface which can be rather rough (by addition of carboxylate brushes), or rather smooth. Both variants sometimes show unspecific binding to proteins. Their surface area available for protein binding is smaller than that of agarose coated beads, therefore they are typically smaller in size (1-2 µm) to provide the same binding capacity.
  • Silica beads are most widely used for nucleic acid purification. They become negatively charged at pH >3, and often show unspecific binding in protein purification.

Size

MagBeads come in different sizes, which has an impact on handling and purification results.
 
  • Small beads (1-10 µm): provide high surface areas available for protein purification, but a smaller magnetic field, which can negatively influence separation, especially from viscous solutions.
  • Medium beads (20-40 µm) : provide efficient separation and large surface area. When combined with agarose surfaces, binding capacities can be in the same range as for small polyvinyl beads.
  • Large beads (70-120 µm): have advantages for special applications, e.g. when purification methods contain both magnetic separation and filtration. When combined with agarose surfaces, binding capacities are still high.

PureCube MagBead Properties

For our standard MagBeads, we chose the following properties to provide optimal results for most applications. However, we can provide a range of customized MagBeads if required.
 
  • Standard: Medium sized beads (20-40 µm) with a ferrimagnetic core, and agarose coating: For high protein binding, low unspecific binding, and efficient separation
  • Custom: Large or extra-large magnetic agarose beads (70-120 µm or up to 400 µm) for special applications are available, and also other commercially available magnetic supports can be modified with your affinity ligand of choice. Contact us to learn more.
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