Immunoprecipitation (IP) is a technique that uses antibodies immobilized on magnetic or agarose beads for isolating a protein from sample material. The target of interest is subsequently detected by Western blot (WB), using labelled secondary antibodies as reporters. This article shares tips for running IP-WB experiments, including how to avoid interference from denatured antibody fragments, to help you achieve publication-quality data.
A typical IP reaction involves combining sample material, beads, and an antibody specific to the protein of interest in a microcentrifuge tube and allowing binding to take effect. A series of washes is then used to remove any unbound material before the protein target is eluted for analysis. Several key factors underpin the success of this process, not least the choice of antibody and solid bead support.
Because IP reactions are used for isolating native proteins, it is essential that primary antibodies recognize the target in its native form. Antibodies that have been validated for IP should be the first choice; where these are unavailable, antibodies that have been successfully used in immunohistochemistry (IHC) are worth a try. By recognizing multiple epitopes, polyclonal antibodies may provide enhanced retention of the target, yet monoclonal antibodies have greater specificity. Whichever antibody is chosen for IP, titration is essential to determine the optimal concentration.
IP was originally developed using agarose beads—porous, non-uniform structures of 50 to 150 μm diameter that benefit from a high surface area for antibody binding. However, because agarose beads are limited by a slow diffusion rate and the need for extensive, manual washing, magnetic beads have become more popular. Magnetic beads are smaller (1 to 4 μm diameter), solid, spherical structures that offer the advantages of fewer, faster washing steps and compatibility with automation.
Many IPs are performed using Protein A or Protein G beads, which bind the antibody Fc region. Yet, since Protein A and Protein G vary in terms of affinity for antibody species and subtype, researchers should consult a binding table when selecting between the different bead types. Alternatively, beads are available conjugated to anti-species antibodies; these function by specifically recognizing and binding the primary antibody used in an IP reaction.
A main challenge faced by researchers performing WB detection of immunoprecipitated samples lies in avoiding interference from denatured antibody fragments, which are derived from the antibody used for target immunoprecipitation. This type of contamination is especially problematic when eluting with a reducing buffer such as Laemmli and can obscure the detection of proteins sharing a similar molecular weight to antibody heavy and light chains (~50 kDa and ~25 kDa, respectively).
One way of eliminating antibody heavy and light chain interference is to use secondary antibodies that recognize only native, non-reduced IgG for Western blot detection. Because these bind the primary antibodies used for Western blotting, without recognizing antibody fragments that have co-eluted with the target, they ensure unhindered detection of the protein of interest for much clearer results.
The main types of reporter used for Western blot detection are horseradish peroxidase (HRP) and a broad range of fluorescent and near-infrared (near-IR) dyes. Choosing between these depends on factors such as the abundance of the target protein, whether there is a need to multiplex, and the availability of suitable instrumentation to measure the assay readout. Where the target protein is scarce, a popular approach is to combine a biotinylated secondary antibody with a labelled avidin or streptavidin conjugate for signal amplification.
Designed as a reporter for IP assays with high specificity and unique detection properties, TrueBlot® secondary antibodies avoid interference from denatured antibody fragments and non-specific binding of other sample contaminants, providing unhindered detection of blotted target bands.
Over the years, researchers have discovered numerous ways for improving IP-WB data. Popular strategies for IP include pre-clearing samples to minimize non-specific binding to the beads; formulating buffers to promote binding, preserve specific affinity interactions, and provide gentler elution; and establishing relevant controls to rule out sources of unwanted background signal. For WB, best practices include using optimized blocking solutions for the chosen detection method; filtering buffers to remove precipitates that could yield unexpected fluorescence; and using rigorously validated antibody reagents that have been confirmed to work in the intended application.
TrueBlot® secondary antibodies eliminate antibody heavy and light chain interference by recognizing only native, non-reduced IgG. Used alone, or in combination with other products from the TrueBlot® range, they ensure publication-quality IP-WB data. Learn more at rockland.com