Working with magnetic beads
Not only are magnetic beads available in different sizes and magnetic properties, they also come with different chemical coatings and functional groups. It is important to choose a magnetic bead that suits your application. Here are a few things to keep in mind while working with magnetic beads.
Understanding the basics of a bead-based experiment
Be clear on what you wish to achieve and know the attributes of your chosen target to enhance binding, sensitivity and specificity of an experiment. Each experiment is different and needs to be empirically optimized.
Handling magnetic beads: The magnetic property of beads determines their functionality. It is good practice to prevent beads from drying out throughout the experiment unless instructed otherwise, as dryness can sometimes crack the bead surface and interfere with elution.
- If required, wash beads in mild buffers before use to remove storage buffer.
- Equilibrate magnetic beads to the solution they will be used in, following the manufacturer’s recommendations to achieve the best results.
- Beads tend to settle out of solution during storage, so resuspend them before use to ensure they are homogeneous. This can be done by gently flicking the tube, maintaining it in a rotating mixer or briefly vortexing. This helps to maintain consistent results across each experiment.
- Be careful not to introduce bubbles at any step during pipetting as beads and sample can be lost.
- Do not to spin down magnetic beads at more than 2000 rpm, as this can change their binding properties and make them difficult to resuspend or bind to targets.
Binding beads to targets: Whether you are purifying antibodies from a crude extract, coating beads with specific antibodies, or binding targets to the beads, it is important to keep them in a rotating mixer at a low speed, around 10-15 rpm. Slow mixing ensures uniform binding and therefore optimized binding capacity. Try using tubes that allow liquid to flow freely to ensure thorough mixing and binding.
Capture: To avoid unwanted loss or carry-over of beads when removing supernatant or eluant, it is important to allow sufficient time for the beads to be pulled out of suspension.
- Allow the tubes containing the suspension to stand on the magnet for at least a minute, and make a visual inspection before removing the liquid fraction.
- Ensure that the tube remains on the magnet during the pipetting process; otherwise the beads may return into solution.
- Avoid disturbing the bead pellet when removing the supernatant.
- To reduce non-specific background signals, carefully remove all the supernatant. This may involve brief air-drying steps.
Washing: When washing the beads, you can reduce non-specific binding and carry-over by increasing either the number of washes, duration of washes or salt or non-ionic detergent (such as tween-20) concentrations. Be sure to use sufficient wash buffer to cover the beads.
Elution: Elute the desired product in a sufficient volume of buffer. Although too great a volume may result in diluted targets, insufficient buffer volume reduces elution efficiency.
Consider your target
The most important aspect of any magnetic bead-based experiment is the target: its source, sample preparation, and purpose in an experiment.
Although magnetic beads allow the freedom to work with samples of varying viscosities, if they are not lysed and homogenized appropriately your results will be inconsistent, no matter how good the protocol is.
- In general, it is better to use stringent buffers for nuclear, organellar or whole cell extractions, and mild buffers for cytoplasmic extractions.
- When working with plant cells, tissue and some Gram-positive bacteria, grinding or bead beating methods should be used to physically disrupt the cells and release the target into the solution. This will facilitate access by the beads for binding.
- If you are working with degradable samples such as proteins and nucleic acids, make sure you use suitable inhibitors.
Performing experimental steps on ice helps to ensure the target remains intact. This will also reduce non-specific background signals from degraded samples. If you intend to use these samples in future experiments, store them at -20˚C or -80˚C (for long-term) with appropriate inhibitors. Do not freeze-thaw your samples more than once.
For low abundance targets, it may be necessary to concentrate the sample prior to undertaking your bead-based experiment. Depending on the target, this can be achieved with a variety of methods including concentration columns, dialysis and re-elution in smaller volumes. Alternatively, some bead-based protocols enable the target to be concentrated as required within the protocol itself. Check the manufacturer’s recommendations for more details.
Optimize and standardize your protocols to achieve the best results
While magnetic beads are versatile and often provide a faster and easier solution than more complex and conventional methods, two important features that require optimization for successful experiments are:
- Bead specificity, the ability to bind target molecules with accuracy
- Binding capacity (the amount of sample bound per bed volume of beads)
Certain protocols might require more optimization, depending on the characteristics of the components and requirements of an experiment. Optimizing and standardizing your protocols will yield the most consistent, reliable results, with greatest efficiency.