Overview of immunoprecipitation with magnetic beads
Immunoprecipitation is a technique that uses antibodies to purify a molecule of interest out of solution. In a general protocol, an antibody against your molecule of interest will be pre-bound to a magnetic bead. The pre-conjugated beads will be mixed with a mixture, such as a cell lysate, and antibodies will bind their specific target in solution. When the mixture with beads is placed in the presence of a magnetic force, using a classical magnetic separator or modern biomagnetic separation system, the beads will be tightly held against the side of their container. Modern magnetic separators have been innovated to provide many sizes of magnets and they overcome challenges with the strength of the force for efficient separation. Modern separators provide a constant force that allows for stable separation over time. With the beads so strongly immobilized, one can simply remove the liquid from the container and replace it with a new clean buffer. When the container is taken out of the magnetic field, the beads will go into solution, still bound to your molecule of interest, now precipitated out of their original solution. This can also be done with free antibodies, which can be mixed with a cell lysate to bind their target, and then are bound to magnetic beads.
Types of Immunoprecipitation with magnetic beads
The overview describes a protocol where a primary antibody is bound to the magnetic bead, for direct binding to target molecules, but there are several other types of bead conjugations with secondary antibodies or surface chemistries that are made for different protocols and budgets. Magnetic beads that are pre-conjugated with protein A, protein G or protein A/G hybrid, can be used to capture many types of antibody subtypes. Charts are available online that can help guide which of the three would best bind the antibody you are using to bind your molecule of interest. Magnetic beads pre-conjugated to secondary antibodies are also available to bind your anti-mouse or anti-rabbit monoclonal antibody in solution. Beads are also made pre-conjugated to streptavidin to bind molecules with a biotin tag. Lastly, there are several types of surface activations on magnetic beads that can help you troubleshoot your immunoprecipitation experiments.
How magnetic beads differ from agarose
Magnetic beads have a different capacity to bind antibodies based on their material properties. Agarose beads are larger, but porous, while magnetic beads have a smooth surface that lends itself to more efficient antibody presentation for better binding of the molecule of interest. The ratio of yield to surface area ends up being better for magnetic beads. Magnetic beads also allow for greater reproducibility because they are all the same size. The use of the magnet to bind beads is the same each time because the force keeping the beads bound to the edge of their container remains the same each time. This makes the samples from magnetic bead immunoprecipitation more pure, while having more high throughput capacity, and potential automation of the process. Overall, magnetic beads make the immunoprecipitation faster and increase yields due to their simplicity of use.
When to use magnetic beads?
Magnetic bead technology is optimal for all types of immunoprecipitation protocols. There are CO-IP protocols, which look for proteins that are co-precipitated. Chromatin-immunoprecipitation, ChIP and ChIP-seq, look for protein interactions with DNA and even sequence the precipitated molecules of interest. Many adaptations have been made to the traditional immunoprecipitation, and they can benefit from the use of magnetic beads.