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Immunoprecipitation (IP) is a technique for purifying small amounts of proteins from biological samples, such as cell lysates or tissue homogenates. It can be used to determine the presence and relative abundance of a single antigen, including its state of post-translational modification. Additionally, IP allows for enriching a target of interest in a complex with its various binding partners, when it is more commonly referred to as co-immunoprecipitation (co-IP), chromatin immunoprecipitation (ChIP), or RNA immunoprecipitation (RIP), depending on the binding partner in question.
During a typical IP reaction, a target-specific antibody is immobilized on agarose or magnetic beads, which are then added to the sample. Following incubation, the beads are harvested by centrifugation or with the use of a magnet, and are washed several times to remove any unbound material. Lastly, the protein is eluted from the beads for analysis, usually by western blotting. A variation of this method involves incubating the antibody with the sample prior to adding the beads, which can help to increase yields if the target of interest is present in low concentrations.
An advantage of using agarose beads for IP is that their highly porous structure maximizes antibody binding. However, agarose beads require long incubation steps (to facilitate antibody diffusion) and have an associated risk of protein damage from centrifuge-based washing. Magnetic beads benefit from shorter incubation times and more gentle wash steps compared to agarose beads, and are compatible with automation. Using magnetic beads also eliminates the need for pre-clearing, which is where the sample and the beads are incubated with an isotype control antibody prior to running the IP reaction to remove sources of non-specific binding.
When selecting antibodies for IP, it is important to look for products that recognize the native protein. If an IP-validated antibody is not available, an antibody that has been proven to work for immunohistochemistry (IHC) or immunocytochemistry (ICC) is often worth a try. In general, polyclonal antibodies are preferred for IP as they can capture more of the antigen due to binding multiple epitopes. Monoclonal antibodies tend to be reserved for downstream western blot analysis due to their high specificity.
SouthernBiotech offers an extensive selection of antibodies for IP, all of which are developed and validated in-house by our highly experienced team. Our product offering includes non-specific mouse, rat, rabbit, and goat antibodies, which are widely cited. Like all of our antibody products, our IP-validated antibodies ensure high specificity and accuracy in target recognition and are rigorously tested to safeguard the reproducibility of your results.
