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Immunoprecipitation (IP) is a method used to enrich or purify your target protein from a complex mixture, such as a cell lysate, using a solid support. After IP you can analyze your samples by various techniques such as Western blot, ELISA or Mass Spectrometry. Some of the most critical factors to consider are the method format, antibody selection, type of support and buffer optimization.

Method Format

There are three options when it comes to performing a classic IP. In the first approach, the antibody against your target is immobilized on a solid support. After immobilization, the support is washed and blocked to remove free antibody and prevent nonspecific binding. The support coupled to your antibody is then incubated with cell lysate containing your target protein. After the sample binding step is complete, the support is washed and the target protein is eluted. Choose this method if you want a high yield of target and control over the antibody binding step.

In the second method, the unbound antibody is incubated with your cell lysate. Once immune complexes are formed, the complexes are captured on a solid support. Choose this technique when the target concentration is very low, binding kinetics for the antibody to antigen are slow or when the antibody’s affinity for the target is weak.

If you are in a rush, you can try to incubate all three components at the same time. Incubation of Protein A/G, antibody and lysate simultaneously may save some time but the yield will probably be the lowest of the three methods.

Antibody Selection

Choosing the right antibody is critical when performing immunoprecipitations. When possible, use an affinity purified polyclonal antibody to capture your target. Since polyclonal antibodies bind to multiple epitopes on your target, the immune complexes will be stronger and you will have a higher rate of retention. Ideally, you should use an antibody from an alternate species for detection by Western blot or ELISA. Using an antibody from an alternate species will aid in data interpretation since you will not detect antibody that was co-eluted with your target. If you only have one antibody and it runs close to the heavy or light chain of your Ab, consider using a Protein A-HRP conjugate for detection in Western blot. Protein A will only recognize the native (non-reduced) antibody, which will eliminate background from your denatured IP antibody (1).

Type of Support?

Agarose is by far the most popular choice due to its durability and minimal nonspecific binding. Protein A or Protein G are typically linked to the support for convenient attachment of your antibody. When using Protein A/G it is important to consider the binding characteristics of your immunoglobulin to select the most suitable protein to use, see Table I below for binding characteristics.

If co-elution of the antibody is a problem, crosslink the antibody to the support with disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3) or directly link your Ab using an activated surface. The use of magnetic beads has been growing in popularity due to their ease of use and increased throughput capabilities.

Binding and Wash Buffers 

Typically, antibody-antigen interactions will occur in any standard buffer at neutral pH, such as PBS or TBS. Most IgG’s will bind to protein A and protein G at a physiological pH and ionic strength. Make sure to check out your product’s user manual as the optimal binding of antibody may be achieved at an alternate pH or ionic strength.

Select a wash buffer that maintains the specific antibody-antigen interaction while minimizing nonspecific binding. It is common to use PBS or TBS with 0.05-1% detergent, such as Tween®-20, Triton™ X-100, NP-40 or CHAPS. Increasing the salt concentration of your wash buffer is another means of increasing the stringency to reduce background. Addition of low levels of reducing agents (1-2mM DTT or β-mercaptoethanol) can help disrupt nonspecific interactions that are mediated by disulfide bridges.

Elution Buffers 

The most effective, non-denaturing elution buffer for immunoprecipitation is 0.1M glycine at pH 2.5-3. Elution at low pH will dissociate most antibody-antigen interactions as well as antibody-Protein A/G interactions if the Ab has not been crosslinked or covalently linked to the support. If the downstream application is SDS-PAGE or Western blot, it may be tempting to elute in reducing sample buffer. Elution with reducing sample buffer is effective but will cause non-specific proteins to co-elute with your target. Fragments of subunits of Protein A/G may also be stripped from the support with a harsh elution.

References

  1. Lal A, Haynes SR, Gorospe M (2005) Clean Western blot signals from immunoprecipitated samples. Mol Cell Probes 19: 385-388. S0890-8508(05)00044-7 [pii];doi: 1016/j.mcp.2005.06.007 PMID:16146684
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