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  • ChIP training
    Part 4: optimization and troubleshooting


    Welcome to our training series on chromatin immunoprecipitation (ChIP). We'll show you ChIP basics and essential protocols before moving on to optimization, troubleshooting, and more advanced techniques.

    In Part 4 of our series on ChIP, we’ll go through optimization and troubleshooting tips that will help you get the most out of your ChIP experiments. We’ll then answer some of the most frequently asked questions (FAQs) on ChIP. Finally, we’ll share some tips for carrying out a ChIP experiment with low cell numbers.


    Part 4 overview

    4.1 Optimizing your ChIP protocol
    4.2 Troubleshooting
    4.3 ChIP tips and FAQs
    4.4 ChIP with low cell numbers


    4.1 Optimizing your ChIP protocol

    Choosing the right ChIP-grade antibody is essential for the success of your ChIP experiment. Not all antibodies are appropriate for ChIP experiments, and many antibodies are not of ChIP quality or validated for ChIP applications. Also, it is critical to include the right controls to ensure that the experiment worked as intended.

    To achieve the best possible results, you may need to make alterations to your protocol or reagents. Here are a few things that may require optimization: cross-linking, chromatin fragmentation, antibody concentration, and wash buffer stringency.

    In this video, you’ll learn how to pick the right antibody, optimize chromatin preparation, and choose the appropriate controls.


    When choosing an antibody for ChIP, pay attention to antibody clonality (ie polyclonal vs monoclonal). Recombinant monoclonal antibodies provide high specificity for their target, low non-specific cross-reactivity, and minimal batch-to-batch variations.

    Watch here to find out how using a monoclonal antibody can ensure reproducibility in your ChIP experiments.


    4.2 Troubleshooting

    All experiments and model systems are different, so sometimes it isn’t enough to follow a standard protocol to get the results you were expecting. If you are having difficulties getting your ChIP experiments exactly how you expect them, look over our troubleshooting tips. In this video, we tackle the most common problems, such as high background in non-specific antibody control, a low resolution with high background across large regions, low signal, and problems with PCR amplification.


    4.3 ChIP tips and FAQs

    Now that you’ve been through all the main steps for ChIP and know how to optimize the protocol, you should have a good idea of what you need to do to get results. Here we share with you some extra tips for different steps of the ChIP protocol, including cross-linking, chromatin fragmentation, and detection.

    Useful tips for a successful ChIP experiment:

    • If ChIP is not an established technique in your lab, you might consider using a kit. Abcam’s ChIP kits will allow you to perform ChIP experiments in 5 hours by using a novel binding technology that eliminates the standard overnight antibody-bead binding step.

    • Always perform a time-course experiment to optimize cross-linking conditions. We would suggest cross-linking the samples for 2–30 min. Add glycine to quench the formaldehyde and terminate the cross-linking reaction.

    • For the chromatin fragmentation step, run a time course every time you set up an experiment.

    • Keep in mind that enzymatic cleavage will not produce random sections of chromatin. Micrococcal nuclease favors certain areas of genome sequence over others and doesn’t digest DNA evenly or equally. Results may not be entirely accurate as certain loci could be over-represented, and some data may be missed.

    • Although sonication is most appropriate for X-ChIP and enzymatic digestion is ineffective on fully cross-linked samples, micrococcal nuclease digestion can be useful when gentle or incomplete cross-linking is required, and it can improve resolution in combination with sonication.

    • Avoid foaming during sonication as it results in a decrease in energy transfer within the solution and will decrease the sonication efficiency.

    • Sonicated chromatin can be snap-frozen in liquid nitrogen and stored at -80°C for up to 2 months. Avoid multiple cycles of freeze-thaw.

    • Certain areas of the genome will purify better than others, and some nucleosomes may re-arrange during enzymatic fragmentation. As a result, it is important to generate PCR primers to several regions in the starting material, as well as the purified/ChIPped material, as controls for spurious results. Generate starting material by lysing the starting cells and take a sample for simple PCR of control regions in parallel with ChIP.

    • With variations in starting material possible, data should always be normalized for the amount of starting material to remove errors introduced due to uneven sample quantities. To normalize your data, take the final amplicon value and divide it by the amplicon value of input material.

    • For histone modifications, the immunoprecipitated material is usually normalized to the input amount and the amount of the relevant immunoprecipitated histone. For example, ChIP with an H3K4me3 antibody will be expressed relative to the input amount and the amount of H3 immunoprecipitated.

    If you are still finding something difficult or have any questions, try to look through our list of ChIP FAQs:

    To cross-link or not to cross-link?
    ChIP for histone modifications is unlikely to require cross-linking, whereas non-histone proteins such as transcription factors and proteins contained in DNA-binding complexes will probably need cross-linking.

    How do I cross-link?
    Use formaldehyde as the links it forms are reversible. UV cross-linking is not appropriate as it is irreversible. Other chemicals like cisplatin can be used to selectively crosslink only between DNA and protein. Dual crosslinking, using formaldehyde and EGS (ethylene glycol bis (succinimidyl succinate)), may be required to study interactions between DNA and particularly large protein/RNA complexes.

    Can I cross-link too much?
    Yes. Cross-linking is a time-critical procedure and should only be carried out for a few minutes. Excessive cross-linking can lead to several issues including a reduction in antigen availability and sonication efficiency. For example, epitopes may be masked or changed, affecting the ability of the antibody to bind the antigen, which in turn causes a reduction in the material in your sample.

    How do I get consistency in my digestions?
    Be sure to aliquot your stock enzyme after purchase and run a new time course with a fresh aliquot every time you set up an experiment. Although enzyme quality may vary over time in storage, the risk of variation within chromatin preparations (degree of compaction, etc) is far higher; one chromatin sample should not be treated as being the same as all others before it. X-ChIP should be carried out as a control experiment when doing N-ChIP to assess any dynamic and unwanted changes resulting from the absence of cross-linking.

    What if I can’t find a ChIP-grade antibody?
    If none are available, then antibodies that work in immunoprecipitation (IP) and immunocytochemistry (ICC) are good candidates. For N-ChIP, we recommend characterizing antibody specificity using peptide competition in western blot. Ideally, specific antibodies for ChIP should be affinity-purified; however, many laboratories use sera as their antibody source and then overcome background problems that may arise with stringent buffers. Antibodies for histone modifications need to be thoroughly tested for specificity, eg by peptide array.

    What antibody controls could I use?
    Histone H3 tri-methyl K4 (H3K4me3) and tri-methyl K9 (H3K9me3) are popular positive controls to use when studying active and inactive genes, respectively. Remember that these antibodies are not positive and negative controls per se, as this will depend on the locus you are studying: if there is no H3K4me3 at the particular locus of interest, the best anti-H3K4me3 ChIP-grade antibody in the world will not immunoprecipitate anything from this region and, therefore, will not be an appropriate positive control.

    As a negative control, use an antibody that recognizes a non-chromatin epitope such as an anti-GFP antibody. Chromatin remodeling may move or remove histones at a particular locus, eg an active promoter, so use a control antibody against a non-modified histone, such as histone H3, to check for the preservation of nucleosomes at particular genomic loci.

    When analyzing histone modifications, you need to normalize to histone content, eg using an anti-H3 antibody.

    The antibody is working for ChIP but the signal is weak—how can I remedy this?
    As a first step, you can try a different type of ChIP. For example, if you are performing N-ChIP, try X-ChIP. Alternatively, look in a different location. It may be that the antigen is present but not on the genome loci that you are looking at. It is a good practice to try different antibodies, when available, to find the one that works best in ChIP. Finally, it might be that the epitope of interest is being masked in X-ChIP: you may need to further optimize the cross-linkage time course.

    What antibody concentration should I use in my ChIP experiment?
    To start with, use 3–5 µg of an antibody for every 25–35 mg of pure monosomes used. If you are doing a quantitative ChIP, then you may need to match the amount of chromatin with the same amount of antibody. As with many techniques, it is essential to optimize the antibody amount at the start.

    What histone control sample should be used for ChIP?
    When immunoprecipitating histone modifications, purified histone H3 and H1 can be used as positive controls for the quality of the experimental histone preparation (histone H1 is usually used for X-ChIP).

    What buffer is recommended?
    The more stringent the buffer used, the better (ie higher concentrations of salt and detergent in the buffer will lead to cleaner results). The buffer must be optimized for every new ChIP experiment as a compromise must be found between low backgrounds and detrimental effects on the target. NP-40 can be used as a detergent and RIPA is also commonly used for X-ChIP.

    What other treatments might affect my ChIP results?
    TSA, butyrate, or colcemid addition do not generally affect ChIP. Some antibodies are affected by relatively low concentrations of SDS. Do not centrifuge sepharose beads at high rpm (do not exceed 6,000 rpm) as this will compact the beads and damage them.


    4.4 ChIP with low cell numbers

    Standard ChIP workflows require a large number of cells. You need approximately 106–107 cells as starting material, below which the assay is hindered by high background binding, poor enrichment efficiencies, and loss of enriched library complexity. However, these large sample sizes can be difficult to obtain, specifically when examining precious sample types like transgenic mouse tissues or clinical samples. To adjust for lower sample inputs, several strategies can be applied.

    1. Improving enrichment efficiency and minimizing sample loss

    Several adjustments to the ChIP workflow can increase enrichment efficiency and minimize sample loss for low input samples.

    • The quality and properties of the sample itself are important considerations. Specifically, in formalin-fixed paraffin-embedded (FFPE) samples, over-cross-linking can cause problems. You may need to use methods to extract soluble chromatin from FFPE samples.

    • The kinetics of the IP with low concentrations of antigen can be optimized by modifying variables like buffer pH, ionic strength, and time of incubation.

    • Broad DNA fragment size distribution hinders the analysis of low-input ChIP, which can be remedied by more limited sonication and/or MNase digestion for more uniform fragmentation.

    • While bacterial DNA is sometimes used as a blocking agent to reduce background for standard ChIP, it is not advised for low-input ChIP as it carries through the assay and confounds data analysis. Other blocking agents such as inert proteins or mRNA can reduce background binding in low-input ChIP, without contaminating the data.

    • Miniaturization of the assay into microwell formats facilitates automation and increases the concentration of the antigen (target transcription factor) during the IP workflow – this avoids the “dilution effect” of low antigen concentrations that favor dissociation of the antibody-antigen complex and decreases the efficiency of ChIP.

    • You can maximize sample retention using single-tube assay formats and magnetic bead purification rather than phenol-based extraction after each assay step.

    • The immobilization of antibody and washes to remove non-antibody bound material is often overlooked. Standard protocols use Protein A/G, but alternatives like epitope-tagged proteins may run the risk of over-expression and introduction of artifacts.

    • High-sensitivity ChIP kits are commercially available for the detection of low cell numbers.

    2. Readout and downstream data processing platforms

    In addition to the assay itself, the choice and optimization of downstream processing (ie sequencing, array, or PCR) and bioinformatic analysis are also important.

    • The detection platform impacts the assay’s sensitivity. ChIP-seq is the gold standard platform for high sensitivity, with consistently lower noise than ChIP-on-chip.

    • The most common issues in low-input ChIP-seq are high numbers of unmappable reads, PCR duplicates, and poor library complexity. Therefore, library preparation must be optimized for low input samples by optimization adapter ligation to avoid amplification-derived error and bias. Maximizing the efficiency of ChIP enrichment, as described above, can also help.

    • Bioinformatic workflows should be adapted to take into account likely process-derived biases in the data.


    Summary

    You should now be fully equipped to tackle a ChIP experiment. You also have a good understanding of:

    • How to choose the right antibodies and controls for a successful ChIP experiment
    • How to optimize your ChIP protocol
    • Where to find help and troubleshooting tips
    • How to optimize ChIP workflow when working with low cell numbers

    And that's the end of the course. We hope you enjoyed it and are better equipped to get the most out of your ChIP experiment.

    If you feel ready, try taking our ChIP quiz to test your new knowledge.

    Be sure to keep an eye on the Abcam training page as our new training series for different applications become available.