ChIP: Immunoprecipitation, washing and elution

Immunoprecipitation

Monoclonal vs. Polyclonal Antibodies?

Selecting an appropriate ChIP antibody is the one of the most critical steps toward a successful ChIP experiment. Even the highest quality antibodies, which may perform very well in typical Western blot validations, may not be suitable for ChIP. It is best to consider only antibodies that have been validated specifically in ChIP. If your antibodies are not specifically quality controlled and proven to perform in ChIP (e.g. ChIPAb+™ validated antibody primer sets) we suggest you evaluate several potential antibodies before selecting one for your actual ChIP experiments.

Below are a few parameters to consider before selecting your antibody. Either monoclonal or polyclonal antibodies will work for ChIP. A monoclonal antibody recognizes a specific epitope on the target protein. Monoclonals provide the advantage of being highly specific with less of a propensity for nonspecific binding. In addition, monoclonal antibodies perform more consistently from batch to batch due low variability in their clonal nature. However, if the epitope recognized by the monoclonal is masked or altered by previous steps in the protocol, such as crosslinking, then monoclonal antibodies will not be effective in isolating your target protein and its associated DNA sequences. Fortunately, this masking rarely affects monoclonal antibodies.

In contrast, polyclonal antibodies recognize multiple epitopes of a target protein. A polyclonal may therefore be more effective even if a few epitopes are masked by crosslinking. However, because polyclonals recognize multiple epitopes, this can increase the probability that nonspecific binding will occur. In addition, it is important to also consider that the specificity of the polyclonal population may drift over time during immunization, unless the serum from which the antibody is purified is pooled prior to preparation or purification. A related point is that most commercial polyclonal antibodies may differ from batch to batch. The degree of variation will depend upon the manufacturing and quality control practices of the vendor. For example, polyclonal antibodies to modifications have finite amounts of serum available and often need to be remade starting with the immunization of a host animal. Consequently, the specificity and affinity of these antibodies can vary from batch to batch. Larger manufacturers of antibodies such as MilliporeSigma are able to address this by immunizing multiple animals followed by screening and pooling of materials demonstrating appropriate affinity and specificity. To ensure consistency, the performance of the final antibody can be compared to previous batches.

Regardless of your choice of monoclonal or polyclonal, when selecting a commercially prepared antibody, for ChIP, the ideal antibody will have data demonstrating specificity as well data showing reliable performance in ChIP and other key applications.

Immunoprecipitation Tips

• Whether you select a monoclonal or polyclonal antibody for your ChIP experiment, you must optimize the dilution of your antibody for your specific analysis. If you use excess antibody, you may succeed in immunoprecipitating your target protein, but you may also observe higher nonspecific binding or reduced specific signal. In contrast if use you use too little antibody you will typically observe low recovery of your target.
• For the best results, ChIP antibodies should be well characterized, proven to bind to its target protein, rigorously tested for specificity, and ideally validated in ChIP.
• Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin Immunoprecipitation. Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state.

Washing the Immunoprecipitate

After immunoprecipitation, the antibodies, beads, and protein A or G often have biomolecules associated with their surfaces that are not related to antigen recognition. It is therefore necessary to perform a series of wash steps with ChIP-specific buffers to remove nonspecific chromatin, protein, and nucleic acids from your immunoprecipitate, because these nonspecific components can significantly increase background signals, produce high variability, and contribute to failure of the ChIP assay. In some cases, multiple buffers (for example, high salt, low salt, lithium chloride, and stringent TE wash), or buffers of increasing stringency, are used to reduce binding of nonspecific molecules.

Other protocols involve simpler buffer systems. Regardless of the wash methods you use, you should use consistent wash conditions: maintain consistent buffer temperatures, wash incubation times, and rotation speeds of any apparatus used for washing. In certain cases, background signals may be reduced by increasing the number of washes, although significant improvement of ChIP signals is ultimately determined by the quality of your ChIP antibody and the nature of your ChIP target.

Elution and Crosslink Reversal

The elution and crosslink reversal steps are necessary to dissociate your chromatin complex from the antibody and beads and to isolate your ChIP’d DNA from the protein portion of the chromatin complex. If you used magnetic beads, elution can be easily done with a magnetic rack and appropriate elution buffers (e.g. sodium carbonate buffers). It is also possible to elute using a peptide competition assay, in which the antibody/protein/DNA complex is incubated with peptides exhibiting greater affinity for the antibody than your target protein, resulting in displacement of your target protein from the antibody complex. This approach may significantly reduce background signals but can be expensive due to the costs of the synthetic peptides.

After elution with agents such as sodium carbonate, and before qPCR analysis, it is critical that you reverse the formaldehyde crosslinks between lysine residues and DNA. Typically, crosslinks are reversed by incubation with proteinase K and heat. You may need to further purify the DNA sample by extraction with a combination of organic solvents, such as phenol:chloroform extraction (see supplementary protocols). Alternatively, purify the DNA from the digested protein/nucleic acid mixture by silica-based chromatography (i.e. spin columns), by magnetic DNA purification particles, or by chelating agents such as Chelex®. We recommend that you perform an RNAse digestion step prior to proteinase K digestion to remove contaminating RNAs from the ChIP reactions.