Tips for Immunoprecipitation

Immunoprecipitation (IP) is a well-established technique used to isolate a specific protein or group of interacting proteins from a complex mixture of many different proteins using an antibody immobilized on a solid support. These solutions are often in the form of a crude lysate of cells, an animal tissue, or a plant. IP is an important step in many proteomic studies designed to explore the presence, relative abundance, protein function, protein-protein interactions, post-translational modifications, and expression profiling of proteins. Purified proteins obtained by immunoprecipitation can be analyzed by a variety of techniques, such as ELISA and Western blotting.

Although the P technique is procedurally simple, the variables affecting the success of any specific experiment are many. IP conditions can be optimized to successfully isolate adequate amounts of specific protein. Consideration of main factors involved in IP can help to identify the components that are most likely to affect particular experiments. Below we describe the various factors that have the largest effect on desired yield and purity of target proteins.

• Lysate Preparation

  The quality of the sample used for immunoprecipitation critically depends on the right lysis buffer. The ideal lysis buffer will stabilize native protein conformation, inhibit enzymatic activity, prevent antibody binding site denaturation, and ensure maximum release of proteins from the cells or tissue for capture and analysis. Non-ionic detergents such as NP-40 and Triton X-100 are less harsh than ionic detergents such as SDS and sodium deoxycholate. Other variables that can affect the success of IP include salt concentration, divalent cation concentration, and pH. Non-denaturing buffers containing non-ionic detergents can be used if the IP antigen is detergent-soluble and the antibody can recognize the native form of the protein. Denaturing buffers, such as radio-immunoprecipitation assay (RIPA) buffer, are more stringent buffers because of the addition of SDS or sodium deoxycholate. While these buffers do not maintain native protein conformation, proteins that are difficult to release, such as nuclear proteins, can be released with denaturing buffers. Detergent-free buffers can also be used if the target protein can be released from cells by physical disruption, such as mechanical homogenization or heat.

  Proteolysis, de-phosphorylation, and denaturation can start as soon as cell lysis occurs, this can be slowed down by keeping the samples on ice or at 4°C at all times and by adding protease and phosphatase inhibitors to the lysis buffer.

• Preclearing Lysates

  Lysates are complex mixtures of proteins, lipids, carbohydrates, and nucleic acids, and thus some amount of non-specific binding to the IP antibody or the beaded support will occur and negatively affect the detection of the immunoprecipitated proteins. In most cases, pre-clearing the lysate by incubating the prepared lysate with the beaded support before commencing immunoprecipitation is a way to remove potentially reactive components that are binding non-specifically to bead components (i.e. coupled secondary antibodies) or the beads themselves. Another pre-clearing technique involves the addition of a non-specific antibody of the same species of origin and isotype as the capture antibody. This process will remove anything that might also bind non-specifically to the capture antibody during immunoprecipitation. The result will be a lowering of background and an improved signal-to-noise ratio.

• Choosing Antibodies

  For immunoprecipitation, the antibody used for purification is an important factor that can affect the yield. Polyclonal antibodies, where possible, should be considered for the capture of a target protein. Polyclonal antibodies bind multiple epitopes on the target protein and form tighter binding immune complexes with higher retention rates.

  The use of antibody pairs, such as a capture antibody from one species, and a detection antibody for Western blotting from another species, is an additional factor to consider for successful immunoprecipitation. In addition to the origination from different species, the antibody selection process should ensure that both antibodies recognize different epitopes of the target protein. A combination of a polyclonal capture antibody and a monoclonal antibody for detection will guarantee maximum capture efficacy with high detection specificity.

• Binding and Wash Buffers

  In most cases, antibody-antigen interactions are fairly robust and will occur in any standard buffer of near-neutral pH, such as phosphate-buffered saline (PBS) or Tris-buffered saline (TBS). Although IgG’s from most species and subclasses bind protein A or protein G near the physiological pH and ionic strength, specific protein A and protein G binding buffers can increase binding. However, such buffers may not always be appropriate for antigen or binding protein interaction. For example, protein A binds IgG best at pH 8.2, while maximum IgG binding occurs with protein G in buffers at pH 5.0.

  When selecting a wash buffer for an IP application, it is important to create conditions in which the desired protein interactions are maintained but non-specific protein binding is prohibited. Ordinarily, the starting point for wash buffer optimization in protein purification methods is either PBS or TBS, which have physiological concentrations of salt and pH levels. Multiple washes (ideally at 4°C) with simple wash buffers such as PBS or TBS either alone or with low detergent concentrations (typically 0.5-1.0% of NP-40, Triton X-100, or CHAPS) or by moderate adjustments to salt concentrations, can be used to reduce background. If non-specific interactions persist and the desired interaction is still strong, the stringency may be further increased by increasing the sodium chloride concentration to 0.5 M or 1 M. Low levels of reducing agents (such as 1-2 mM DTT or β mercaptoethanol) can help disrupt non-specific interactions mediated by disulfide bridges.

• Elution Buffers

  To ensure that the target protein is eluted from the beads, elution buffer at the correct strength and pH must be chosen for the elution of proteins. For analysis of immunoprecipitated proteins by reducing SDS-PAGE and Western detection, elution of proteins directly in reducing SDS-PAGE sample buffer would be ideal. This buffer, designed to denature and reduce proteins for electrophoresis, is very effective in dissociating the affinity interactions. However, elution in SDS-PAGE sample buffer will cause multiple non-specific proteins to co-elute with the antigen. Fragments of the immobilized solid support (e.g., subunits of protein A/G) may be stripped from the beads with harsh elution buffers. In such cases, elution in a milder buffer (0.1 M glycine, pH 2.5) and neutralizing before loading to SDS-PAGE gel, will prevent this contamination. The low pH condition dissociates most antibody-antigen interactions as well as the antibody-protein A/G interactions.

• Choosing Secondary Antibodies

  Secondary antibodies that recognize the heavy and light chain of the primary antibody for Western blot detection of IP samples will always result in two bands (the heavy chain at 50kDa and the light chain at 25kDa). It is therefore difficult to detect the protein of interest if it migrates around either the 50kDa or 25kDa markers. To avoid interference by the antibody chains, we recommend using Trueblot®, which only recognizes primary antibodies in their native (non-reduced) state, and thus eliminates the detection of the denatured primary heavy and/or light chains during Western blotting.