Disclaimer

Please note that this procedure is intended only as a guideline. Please keep in mind that Creative Biolabs cannot guarantee specific ChIP results.

Chromatin immunoprecipitation (ChIP) is a valuable technique for assessing the binding of specific proteins to particular DNA sequences. Creative Biolabs has summarized the research experience to help researchers successfully perform the ChIP experiments. Additionally, Creative Biolabs provides protein-nucleic acid interaction assay services to measure the quantitative parameters of the described binding.

Principle of ChIP

ChIP selectively enriches chromatin fractions that contain specific proteins by using antibodies to immunoprecipitate the protein of interest and its associated DNA. This fraction is then recovered and analyzed by PCR, microarray, or sequencing to determine the genomic locus at which the protein binds.

Advantages of ChIP

ChIP allows researchers to investigate the spatial and temporal dynamics of chromatin interactions and their associated factors. This technique allows us to map minute-by-minute changes in individual promoters or track individual transcription factors throughout the genome (Note that the output is averaged by analyzing cell populations).

ChIP is versatile and provides insights into gene regulation in their natural environment. Euchromatin, which contains active genes and maintains an open and expanded structure, is the most commonly studied chromatin type for ChIP, playing a critical role in transcription, DNA repair, and gene replication.

Procedure of ChIP

ChIP has four main steps to determine whether a given protein binds to a specific DNA sequence in vivo.

• Isolation of total chromatin.
• Chromatin fragmentation to achieve resolution.
• Immunoprecipitation of the resulting chromatin fraction.
• Analysis of the immunoprecipitated fraction to determine the amount of target DNA sequence (or sequences) relative to its abundance in the input chromatin.

Tips for ChIP

Cross-linking

Cross-linking is primarily performed prior to analysis to stabilize DNA-protein interactions. ChIP can be performed in two different ways, depending on whether cross-linking is required: Cross-linked ChiP (X-ChIP), when cross-linking is necessary; Native ChIP (N-ChIP), when cross-linking is not required.

How can I tell if I need to cross-link?

Cross-linking anchors the antigen of interest to its chromatin binding site, making it unnecessary for histones that are already tightly bound to DNA. However, DNA-binding proteins with weak affinity for DNA or histones may require cross-linking to hold them in place and avoid protein dissociation from the chromatin binding site.

The farther the interaction you are interested in is from the DNA, the less effective the ChIP is without cross-linking.

How can I cross-link?

Reversible cross-linking can be achieved using formaldehyde. UV cross-linking is irreversible. Formaldehyde is generally chosen for cross-linking.

Although formaldehyde is a very good DNA-protein cross-linker, it is not effective for protein-protein cross-linking due to its small size (2 Å). It is therefore often difficult to perform microarray analysis of proteins that do not bind DNA directly.

Cross-linking is a time-critical process that should only be performed for a few minutes. Excessive cross-linking can lead to reduced antigen availability and sonication efficiency, such as masked or altered epitopes that can affect the ability of the antibody to bind the antigen and in turn lead to a reduction of material in the sample.

Time-course experiments are always performed to optimize cross-linking conditions. We recommend that samples be cross-linked for 2-30 min and the cross-linking reaction should be terminated by adding glycine to quench formaldehyde.

To further aid in DNA purification, cross-links between protein and DNA are disrupted by treatment with proteinase K, which clears peptide bonds adjacent to the carboxyl groups of aliphatic and aromatic amino acids.

Chromatin Fragmentation

Fragmentation of chromatin is necessary to enable antibody reagents to interact with it. Chromatin can be fragmented using either sonication or digestion with micrococcal nuclease. The appropriate method should be chosen based on the specific trial protocol. Regardless of the method used, a fragmented time course should be run every time an experiment is set up.

N-ChIP with Enzymatic Digestion

Enzymatic digestion using micrococcal nuclease should be sufficient to fragment the sample for N-ChIP. Unlike other methods, N-ChIP does not require cross-linking, which has no potential effect on enzymes that enter its target.

Enzymatic techniques can produce single monomers, about 175 base pairs, providing the highest resolution in standard ChIP. However, certain chromatin binders, such as transcription factors, frequently bind internucleosomal DNA, and purified single nucleosomes are not suitable.

Nucleosomes are dynamic and not cross-linked, and they may rearrange during enzymatic digestion. This can be a problem if mapping genomic regions is necessary, and appropriate controls should be used to monitor any changes.

Enzymatic cleavage does not produce random sections of chromatin. Micrococcal nucleases favor certain regions of the genome sequence and do not digest DNA evenly or equally. Therefore, some loci may be overrepresented, and some data may be missed, leading to potentially inaccurate results.

How Can I Keep My Digestion Consistent?

Stock enzyme should be aliquoted, and a new time course should be run with fresh aliquots each time an experiment is set up. Although enzyme quality may vary with storage time, the risk of variation (degree of compaction, etc.) is much higher in chromatin preparations. Therefore, a chromatin sample should not be treated as identical to all other samples before it.

X-ChIP and Sonication

Formaldehyde cross-linking restricts enzymes such as micrococcal nucleases from accessing their targets. Therefore, sonication is necessary for X-ChIP, and enzymatic digestion is often inefficient for cross-linked samples.

Sonication generally produces DNA fragments of random size and no part of the genome is preferentially cleaved, although this is rarely observed in practice. Fragments produced by sonication averaged 500 to 700 base pairs (2-3 nucleosomes) and were generally larger than those produced by enzymatic cleavage. The size of the fragments created directly affects the resolution of the ChIP program, and fragments up to 1.5 kb are well resolved in ChIP for most purposes.

Although sonication is best suited for X-ChIP and enzymatic digestion is ineffective for fully cross-linked samples, micrococcal nuclease digestion is useful when mild or incomplete cross-linking is required and can be used in combination with sonication to improve resolution.

Foaming should be avoided as it leads to reduced energy transfer within the solution and can reduce sonication efficiency.

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

Antibodies for ChIP

Antibodies are utilized in ChIP to capture proteins and interacting DNA. Ideally, the antibodies used should be fully characterized and their functionality in ChIP should be confirmed. If possible, use antibodies that have been fully characterized and labeled as ChIP grade, or customize antibodies to meet specific experimental needs.

For N-ChIP, peptide competition in western blotting is recommended to characterize antibody specificity. Ideally, Chip-specific antibodies should be affinity purified. However, many laboratories use serum as a source of antibodies and overcome any background issues that may arise from strict buffers.

Even if an antibody is fully characterized, it may not necessarily be functional in X-ChIP due to the effects of cross-linking, which can generate different epitopes and result in the loss of specific epitopes. To test whether an uncharacterized antibody can ChIP, perform ChIP with the antibody and subsequently perform Western blotting with the same antibody.

What if there are no chip-grade antibodies available?

In such case, antibodies that act in IP and IHC are good candidates. For N-ChIP, peptide competition in Western blotting is recommended to characterize antibody specificity. Ideally, chip-specific antibodies should be affinity purified. However, many laboratories use the serum as a source of antibodies and overcome any background issues that may arise from strict buffers. Antibodies used for histone modification need to be thoroughly tested for specificity, for example by using peptide arrays.

Monoclonal antibodies recognize only one epitope, whereas, within a polyclonal antibody population, there will be many antibodies that recognize different epitopes. Polyclonal populations will reduce the probability that all specific epitopes will be masked by the cross-linking process, and thus X-ChIP has a better chance of positive results. However, monoclonal antibodies generally have high inter-assay consistency. For more information on custom monoclonal antibody services, please click to visit.

How do I remedy an antibody that works against ChIP, but has a weak signal?

First, try different types of ChIP. For example, if you are using N-ChIP, try X-ChIP instead. It's also possible that the antigen is present but not at the genomic locus you are examining. In such cases, try using different antibodies, if available, to find the best fit for ChIP. Finally, it's possible that epitopes of interest may be masked in X-ChIP, requiring further optimization of the cross-linking time course.

How do I determine the concentration of antibodies in the ChIP assay?

First, use 3-5 μg of antibody for every 25-35 mg of pure monomer used. If you're doing quantitative ChIP, eventually, you may need to match the amount of chromatin to the same amount of antibody. It's essential to optimize the amount of antibody used at the beginning, if possible.

However, even if the antibody is able to immunoprecipitate the target protein in formaldehyde-fixed chromatin, it doesn't necessarily mean that the ChIP assay has worked because the protein of interest may not be cross-linked to the DNA.

If you observed high background, additional washing may be required. Alternatively, sonicated chromatin can be precleared by incubation with protein A/G beads for 1 hour before immunoprecipitation. This step helps remove any nonspecific binding to the magnetic beads.

Detection

Controls for quantitative PCR

Since some regions of the genome will be better purified than others, and some nucleosomes may rearrange during the enzyme fragmentation, generate PCR primers in multiple regions of the starting material and the purified/microarray material. This helps control for spurious results. To generate starting material, lyse starting cells and sample them in parallel with ChIP for simple PCR of the control region.

Data

Normalize the data for the starting material amount since the starting material may vary. To do this, take the final amplicon value and divide it by the amplicon value of the input material. For histone modifications, normalize the immunoprecipitated material to the input and the amount of the relevant immunoprecipitated histone. For example, a ChIP with an H3K4me3 antibody should be expressed relative to the input and H3 immunoprecipitation amounts. Measuring the amount and quality of the starting material is crucial for the effective interpretation of results.

Other Frequently Asked Questions

Which histone control sample should be used in ChIP experiments?

Calf thymus histone preparation is used as a positive control protein sample to check antibody specificity in western blotting. When immunoprecipitating histone modifications, purified histones H3 and H1 can be used as a positive control for the quality of protein preparation in the experimental group (histone H1 is commonly used in X-ChIP).

Which buffer is recommended?

The more stringent the buffer used, the better (i.e., the higher the salt and detergent concentration in the buffer, the cleaner the result). Optimize the buffer for each new ChIP experiment because a compromise must be found between low background and adverse effects on the target. NP-40 can be used as a detergent, and RIPA is also commonly used in X-ChIP.

What other treatments might affect my ChIP results?

1. Adding TSA, butyrate, or colcemid usually does not affect ChIP.
2. Do not centrifuge sepharose beads at high speeds (no more than 6,000 rpm), which can compact magnetic beads and damage them.
3. Some antibodies are affected by relatively low concentrations of SDS.

Advantages and Disadvantages of N-ChIP & X-ChIP

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