Chromatin immunoprecipitation (ChIP) remains a powerful method to characterize in vivo binding of DNA hugging proteins like histones and transcription factors and more recently has been used effectively for DNA methylation studies. The ChIP technique has been around since the days of big hair and “Miami Vice,” so we won’t review it here much, but the process generally involves these basic steps:
- Isolation of total chromatin or methylated DNA from cells (sometimes after cross-linking to fix the protein to the chromatin)
- Fragmentation
- Immunoprecipitation (IP) of the protein or mark of interest with a specific antibody to isolate regions of interest
- Analysis of the immunoprecipitated DNA to reveal the sequence(s) bound by the studied protein.
In the old days (mid-to-late 1990s), most researchers were limited to using quantitative PCR, or other lower multiplexing analysis to identify DNA sites bound by their protein, but more recently, sophisticated readout technologies like high-density microarray hybridization (ChIP-Chip) and deep sequencing (ChIP-Seq) have boosted ChIP experimental data to levels researchers couldn’t even dream of a few years back. This dream can turn into a nightmare quickly if care is not taken on the front end of these experiments.
ChIP Antibody Selection Questions for the Panel
Antibody selection continues to be one of the most important factor governing the success or failure of a ChIP experiment, so Epigenie asked five of the antibody industry’s leading experts to discuss key considerations when choosing antibodies for ChIP. Please note: no industry experts were harmed during the production of this article.
What are the best guidelines for selecting an ideal antibody for ChIP applications, particularly when the downstream technology will be high-density arrays or deep sequencing?
Karen Halls, Ph.D., Sr Tech Specialist and Rachel Imoberdorf, Ph.D., Sr Characterization Scientist
The antibody should be fully characterized and specific to the target. If an antibody works in IP or immunohistochemistry of fixed samples, there is a high probability it will also work in ChIP. Polyclonal antibodies have a higher rate of success in ChIP than monoclonals because polyclonals consist of a pool of antibodies against different epitopes. For genome-wide analysis, however, it is advisable to choose monoclonal antibodies whenever possible because the batch-to-batch variation is lower.
Jeff Falk, Ph.D., Director of Technology & Business Applications, Aviva Systems Biology
Identifying the appropriate antibody is the key step in deriving high-quality data for ChIP-based array or sequencing studies. We’ve found that generally only 1 in 5 antibodies that work well for western blots also work efficiently for ChIP. The best way to identify appropriate “ChIP-grade” antibodies is to perform validation studies on a large number of antibodies with specificities to multiple epitopes of the factor in question. To address this need, Aviva Systems Biology has made multi-epitope peptide antibodies to the entire family of human transcription factors and co-factors, which enables us to perform validation studies and identify a “ChIP-grade” antibody for virtually any paradigm of interest.
John Heyman, Ph.D., Director of Scientific Development, Active Motif
If you’re going to be spending a lot of time on data analysis from high-density arrays or deep sequencing, you really need to validate your antibody—you want to make sure that the antibody is specific and not pulling down DNA through non-specific interactions.
Sujay Singh, Ph.D., President and CEO, Imgenex Corporation
The antibody should not bind non-specifically to other proteins and should have good affinity for the target protein. Antibodies with the IgM isotype are not good candidates because mouse and rat antibodies of this isotype do not bind to Protein A/G, which is often used to precipitate protein−chromatin complexes.
John Rosenfeld, Ph.D., Manager II, Chromatin Biology, Millipore
At a minimum, you need to know (preferably by a variety of methods) that your candidate antibody is highly specific and selective. At Millipore, we have the ability to validate our antibodies using peptide-coupled beads in the Luminex platform, so that gives us some idea of epitope specificity for particularly challenging epitopes such as histone post-translational modifications. But in the end, you’d better have some additional biological validation that gives you confidence that your antibody does not cross-react with related conformational epitopes within a cell.
How are antibodies validated for ChIP?
John Heyman, Ph.D., Director of Scientific Development, Active Motif
Ideally, you’ll have some induction conditions that you can use in conjunction with a few DNA target sequences that you know will be bound by your protein of interest upon induction. By PCR analysis, you’ll be able to convince yourself that the antibody has really low background and high specificity.
John Rosenfeld, Ph.D., Manager II, Chromatin Biology, Millipore
At Millipore, we have developed an antibody-specific assay to assess an antibody’s suitability for ChIP. Most often, we search the scientific literature to identify published assays demonstrating that a particular antibody target is present in a particular chromatin context. Then, we assess the antibody’s ability to specifically pull out the expected chromatin locus. If the antibody is successful, we challenge the antibody with biological validation, which we believe is critically important. We face some challenges when we lack awareness of the function of the antibody target. In that case, we try to employ the best possible controls, such as chromatin derived from a knockout mouse cell line versus wild-type chromatin.
Sujay Singh, Ph.D., President and CEO, Imgenex Corporation
Antibodies that work well with regular IP assays have a better chance of working in ChIP assays. The antibody should be tested on multiple independent preparations of chromatin.
Karen Halls, Ph.D., Sr Tech Specialist and Rachel Imoberdorf, Ph.D., Sr Characterization Scientist
To validate antibodies for ChIP, it is imperative to know a region in the genome that is bound by the target protein and a region where the target protein is absent. A ChIP experiment using standard conditions is performed. Depending on the results, conditions are then changed, e.g., if there is a very low signal, the antibody concentration and the salt concentration in the final wash can be titrated. It is worth testing the antibody in a standard IP first because an antibody that does not work in IP will not work in ChIP.
Jeff Falk, Ph.D., Director of Technology & Business Applications, Aviva Systems Biology
The best ChIP-validation method is using ChIP−quantitative PCR (ChIP-qPCR) with positive and negative control regions to demonstrate that the antibody exhibits appropriate binding characteristics. Aviva Systems Biology has developed a comprehensive tiling ChIP-qPCR assay that enables us to quickly ascertain whether the antibody in question is capturing factor-specific interactions and is suitable for ChIP-based studies. We have also developed a fully automated, high-throughput antibody validation system that can quickly and efficiently validate antibodies for ChIP with a throughput of 100 targets/week. Obviously, this validation scheme is dependent on having existing knowledge regarding the activity of the factor and the appropriate controls.
Many companies are now selling “ChIP-grade” antibodies. What exactly does this mean at your company?
Jeff Falk, Ph.D., Director of Technology & Business Applications, Aviva Systems Biology
Aviva Systems Biology has an active internal program for validating antibodies and has established a set of “ChIP-grade” antibodies that we know perform well in ChIP-based studies involving specific cell types. These validated antibodies are used extensively by Aviva for our ChIP-DSL and ChIP-Seq service programs in which we provide complete ChIP-based services for customers. These antibodies have not been released to the public as ChIP-grade at this time, but they are being incorporated into ChIP-qPCR kits that will be available soon.
John Heyman, Ph.D., Director of Scientific Development, Active Motif
For us, “ChIP-grade” means that either we’ve done the research in-house to show an antibody works for ChIP, or a customer has reported that one of our antibodies works for ChIP. Even if you have an antibody that has been validated for ChIP, you still need to do a few experiments to show that it’s working in your system. For example, different methods of chromatin preparation or fixation can affect the ChIP results. The advantage with ChIP-grade antibodies is that you know that there’s a good chance they will work. With some antibodies, you just don’t have a chance with ChIP, period.
Sujay Singh, Ph.D., President and CEO, Imgenex Corporation
ChIP-grade means that these antibodies are validated to work in ChIP assays either by us or by our customers. An antibody may work beautifully in western blots, immunohistochemistry, ELISA, and other applications but may not immunoprecipitate the protein when it is complexed with chromatin.
Karen Halls, Ph.D., Sr Tech Specialist and Rachel Imoberdorf, Ph.D., Sr Characterization Scientist
The ChIP-grade antibodies we stock have been tested in ChIP and are covered by our Abpromise guarantee, which offers technical support and refund or replacement if an antibody does not work after optimization.
John Rosenfeld, Ph.D., Manager II, Chromatin Biology, Millipore
We are moving toward segregating our “ChIP-grade” antibodies into a kit-type product line named “ChIPAb+,” in which the antibodies are validated on a lot-to-lot basis in a particular quantitative ChIP assay. This new product includes not only measured amounts of the antibody in ChIP reaction units, but also a negative control antibody for the ChIP assay and aliquots of qPCR primer sets to validate the performance of that antibody lot. When remaking polyclonal antibodies, there is always a risk that not every lot of antibody will work every time. Thus, unless we’ve tested the antibody in ChIP ourselves, every single lot, we don’t promote it as a ChIPAb+ product.
Do ChIP conditions need to be optimized for each antibody? If so, what are the best approaches for optimization?
John Rosenfeld, Ph.D., Manager II, Chromatin Biology, Millipore
Unfortunately, each ChIP experiment is different depending on the source of chromatin, effectiveness of the antibody in ChIP, and relative abundance of the targeted epitope. We recommend that our customers assess the fragmentation of their substrate chromatin preps for each experiment. For our ChIPAb+ antibodies, we’ve tried to reduce the amount of ChIP antibody-related optimization required by presenting a successful experiment with a given quantity of antibody and chromatin. The quantity of antibody may still require adjustment for different chromatin samples, but at least the customer has a starting point for comparison.
Karen Halls, Ph.D., Sr Tech Specialist and Rachel Imoberdorf, Ph.D., Sr Characterization Scientist
The amount of antibody used in the IP should be optimized, as well as the wash steps. Different salt concentrations for the final wash step should be tested (0−500 mM NaCl/LiCl). The sonication and cross-linking steps should also be optimized as these will vary among cell types.
Sujay Singh, Ph.D., President and CEO, Imgenex Corporation
Yes, a good ChIP assay needs two important components: good chromatin preparation and a good antibody. The common methods of chromatin preparation are sonication and enzymatic digestion. The antibodies should be tested on multiple preparations of chromatin prepared by both methods.
Jeff Falk, Ph.D., Director of Technology & Business Applications, Aviva Systems Biology
Several aspects of the ChIP assay need to be optimized including the fixation time and the sonication time for chromatin shearing. Incomplete chromatin shearing is a common source of false positives in ChIP-based reactions. The specificity of ChIP assays can be enhanced dramatically by gel purification of the sheared chromatin in the 300−800-bp range. However, once again the most critical optimization factor is choosing an appropriate antibody that has been validated to efficiently capture factor-specific binding sites.
John Heyman, Ph.D., Director of Scientific Development, Active Motif
Yes, ChIP results are often improved through optimization of chromatin fixation and shearing. For example, sometimes short fixation (2−5 minutes with 1% formaldehyde) can give much better results than the typical fixation (10 minutes with 1% formaldehyde). When possible, cell type and cell culturing conditions should be chosen so that positive and negative control chromatin can be prepared. This allows accurate interpretation of experiments to optimize ChIP conditions and ChIP PCR primers. Active Motif sells ChIP-validated sheared chromatin, as well as ChIP kits. These quality-controlled reagents can increase your confidence in decisions about the antibody.