Purification is a common procedure in most molecular biology experiments, such as the removal of short primers, unincorporated dNTPs, enzymes, and salts from PCR reactions and other impurities. There are various types of purification methods, including filtration, centrifugation and separation. Separation has become a very popular method in life sciences and various types of solid phase support are used. This includes agarose beads, sepharose beads, silica beads, and magnetic beads. Separation using magnetic beads is the quickest, cleanest and most efficient technique out of all the bead separation methods.
Magnetic beads can be coated with specific affinity ligands for antigens, antibodies, proteins or nucleic acids. After the magnetic beads are incubated with the sample, magnetic force is applied to the beads (and attached biomolecules) in order to collect the beads into a concentrated pellet. Once separation is completed, the supernatant can be easily removed and the biomolecule bound to the beads can be eluted from the magnetic beads.
The ability to manipulate the magnetic beads using magnetic force makes the washing steps easier and faster than using agarose, sepharose or silica beads. The purified product can also be recovered in a more concentrated form using magnetic separation method.
Here are some tips for new users:
1. Resuspend your beads thoroughly
Magnetic beads need to have enough magnetic contents to allow simple pull down by a magnet. Conventional magnetic beads have sizes above 1 µm (1 µm = 1000nm). Newer beads are developed with much smaller sizes (down to a couple hundreds of nanometers) to increase beads surface area. This results in increased binding capacity and improved dispersion. Magnetic beads are heavy particles comprised of iron oxide so they sediment over time. The larger the size of the beads are, the faster they sediment. It is important to vortex and thoroughly resuspend the magnetic beads before use to redisperse the beads and to ensure consistency between the aliquots.
2. Wash your beads
Increase the number of washing steps helps to reduce non-specific binding to the beads.When washing the magnetic beads with either ethanol (for nucleic purification) or wash buffer, use enough wash solution to cover the pellet.
3. Know the properties of your beads
Magnetic beads come in different sizes, shapes, magnetic response, coatings, buffer conditions, and functional groups that are attached to them. These variables affect the properties of the beads. It is important to get the basic information of the beads to better understand how to handle them.
4. Capture the beads
Generally the magnetic beads are attracted to the magnet and form a pellet within a minute. Prolong the attraction of the magnetic beads to the magnet to ensure all beads are recovered.
5. Do not disturb the bead pellet when removing the wash solution
When removing the wash solution or supernatant, angle the pipette tip so that the tip does not touch the pellet of magnetic beads. This prevents re-mixing of beads with supernatant. Use a magnetic rack with a slanted side so the bead pellet is concentrated on one side of the tube’s wall. This makes it much easier to remove the supernatant.