Disscussion of ChIP protocol and technique

Supplement your protocols - useful tips for chromatin IP compiled by Dr. Andy Bannister and the Abcam Chromatin Team.

Contents


Why is ChIP such a useful technique?

ChIP dissects the spatial and temporal dynamics and interactions of chromatin and its associated factors. This technique allows us to map minute-by-minute changes at a single promoter, or alternatively, follow a single transcription factor over the entire human genome. ChIP is very versatile and can give us significant insight into how genes are regulated in their natural context.

The principle of ChIP is simple: the selective enrichment of a chromatin fraction containing a specific antigen. Antibodies that recognize a protein or protein modification of interest can be used to determine the relative abundance of that antigen at one or more locations (loci) in the genome. The most commonly ‘chipped’ chromatin is euchromatin. This material contains active genes and maintains an open and extended structure in order to play an important role in transcription, DNA repair and gene replication. By contrast heterochromatin, which contains many inactive genes, is difficult to analyze by ChIP, not least because of its condensed state and generally repetitive DNA sequence.

Here we provide some further insights into the ChIP technique to compliment our X-ChIP and N-ChIP protocols, provided by collaborators and the Abcam team, which we hope will help you troubleshoot this powerful method.


What is ChIP?

ChIP is used to determine whether a given protein binds to a specific DNA sequence in vivo. The ChIP procedure consists of the following steps:

  1. Isolation of total chromatin
  2. Fragmentation of the chromatin (to achieve resolution)
  3. Immunoprecipitation of the resulting chromatin fragments
  4. Analysis of the immunoprecipitated fraction to determine the amount of a target DNA sequence (or  sequences) relative to its abundance in the input chromatin.

(For a full literature review of the technique in its various forms read our ChIP article written by Dr. Graeme Cuthbert and Dr. Andy Bannister, University of Cambridge.)


Cross-linking

Cross-linking of DNA and proteins is often required to stabilise their interactions before analysis. ChIP can be performed in two different ways depending on whether you opt to cross-link your chromatin sample: if you cross-link your sample then the technique is termed “X-ChIP,” otherwise it is referred to as “N-ChIP.”

To cross-link or not to cross-link?

The aim of cross-linking is to fix the antigen of interest to its chromatin binding site. Histones themselves generally do not need to be cross-linked, as they are already tightly associated with the DNA.

Other DNA binding proteins that have a weaker affinity for DNA or histones may need to be cross-linked. This holds them in place and avoids protein dissociating from the chromatin binding site.

Tip: The further away from the DNA your interaction of interest lies then the less effective ChIP will be without cross-linking. E.g. 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. See our N-ChIP and X-ChIP protocols for details. UV cross-linking is not appropriate as it is irreversible.

Can I cross-link too much?

Yes. Cross-linking is a time-critical procedure. Cross-linking should generally only be carried out for a few minutes. Excessive cross-linking can lead to a decrease in the amount of protein bound to the DNA, reduction in the availability of epitopes/changes in epitopes for antibody binding and, in turn, reductions in the material bound/antigen availability in your sample.

Tip: always perform a time-course experiment to optimize cross-linking conditions.


Chromatin fragmentation

Fragmentation of the chromatin is required to make interactions accessible to antibody reagents. To fragment chromatin, you can either sonicate it or digest it with using micrococcal nuclease. Which method you choose will largely depend upon the type of ChIP experiment being performed.

N-ChIP with enzymatic digestion

Enzymatic digestion, with micrococcal nuclease, should be sufficient to fragment your sample for performing N-ChIP. N-ChIP does not call for cross-linking and so there will be no potential affects on the enzyme accessing its target. Using the enzymatic technique it is possible to generate single monosomes (~175 base pairs), providing the highest resolution in ChIP.

Nucleosomes are dynamic and without cross-linking they may rearrange during the enzymatic digestion. This is a potential problem if you wish to map areas of the genome, and suitable controls must be used to monitor any changes (see Detection — controls for quantitative PCR).

Tip: Enzymatic cleavage will not produce “random” sections of chromatin. Miccrococcal nuclease favors certain areas of genome sequence over others and will not digest DNA evenly or equally. Results may not be entirely accurate as certain loci could be over represented and some data may be missed.

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.

Tip: 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.

X-ChIP and sonication

Typically, sonication is necessary for X-ChIP as formaldehyde cross-linking restricts the access of enzymes such as micrococcal nuclease to their targets, meaning that enzymatic digestion will normally be inefficient on cross-linked samples.

Sonication is generally believed to create randomly sized DNA fragments, with no section of the genome being preferentially cleaved. The fragments created by sonicating, which average 500–700 base pairs (2–3 nucleosomes), are typically larger than those created via enzymatic cleavage. The size of the fragments that are created directly affects the resolution of the ChIP procedure; fragments up to 1.5 kb resolve well for most purposes in ChIP.

Tip: 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/incomplete cross-linking is carried out and it can improve resolution in combination with sonication.


Antibodies for ChIP

Antibodies are used in ChIP to capture proteins and the interacting DNA.

Antibodies used for ChIP should ideally be fully characterized

Characterizing antibody specificity using peptide competition in western blot is recommended for N-ChIP. 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 (see other frequently asked questions).

Be aware: even full characterization will not tell you whether or not an antibody will function in X-ChIP, as the effects of cross-linking can be dramatic to the extent that different epitopes may be generated and specific epitopes may be lost.

A polyclonal antibody is preferable to a monoclonal

Whereas monoclonal antibodies recognize only a single epitope, within a polyclonal antibody population there will be a number of antibodies that recognize different epitopes. A polyclonal population will reduce the probability that all specific epitopes will be masked by the process of cross-linking, so there is a better chance of a positive result in X-ChIP.

Don’t have a ChIP-grade antibody?

In general, if an antibody works in normal immunoprecipitation then it is a good candidate for success in ChIP.

What antibody controls could I use?

As a positive antibody control for the technique, Histone H3 (tri methyl K4) is a popular positive control to use when chipping active genes. As a negative control, use an antibody that recognizes a non-chromatin epitope such as an anti-GFP antibody.

Tip: Chromatin remodeling may move or remove histones at a particular locus e.g. 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.

Caution: Remember that these antibodies are not positive and negative controls for the success of the ChIP experiment per se — this depends on the locus you are studying. For example, if there is no Histone H3 tri methyl K4 at the particular locus of interest, then even if you have the best ChIP antibody in the world it will not immunoprecipitate anything from this region and so will not be an appropriate positive control.

The antibody is working for ChIP but the signal is weak — how can I remedy this?

  • Try a different type of ChIP. For example, if you are performing N-ChIP try X-ChIP.
  • Look in a different location. It may be that the antigen is present but not on the section of the genome that you are looking at.
  • Use a different antibody if one is available.
  • Is the epitope being masked in X-ChIP? It may be necessary to further optimize the cross-linkage time course.

What concentration of antibody should I use in my ChIP experiment?

To start with, use 3–5 mg of antibody for every 25–35 mg of pure monosomes used. If you are doing a quantitative ChIP then ultimately 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 amount of antibody at the start if possible.


Detection

Controls for quantitative PCR

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.

Data

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.

Tip: Measuring the amounts (and quality) of starting material is the key to interpreting your results effectively.


Other frequently asked questions

What histone control sample should be used for ChIP?

Calf thymus histone preparation should be used as a positive control histone sample for checking antibody specificity in western blot. When chipping for 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 (i.e., higher concentrations of salt and detergent in the buffer will lead to cleaner results). It is essential that the buffer is optimized for every new chipping experiment as a compromise must be found between low backgrounds and detrimental effects on the target. NP40 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.


Summary of advantages and disadvantages

Advantages

Disadvantages

N-ChIP

Predictable and testable antibody specificity

Not useful for non-histone proteins

Efficient precipitation of DNA and protein

Selective nuclease digestion may bias input chromatin

High resolution (175 bp/monosomes)

Nucleosomes may rearrange during digestion

   

X-ChIP

Good for non-histone proteins binding weakly or indirectly to DNA

May be inefficient antibody binding due to epitope disruption

 

Cross-linking minimizes nucleosome rearrangements

Fixes transient (artifactual) interactions to give a false picture of steady state levels

Good for organisms where native chromatin is difficult to prepare (e.g., yeasts)

Lower resolution chromatin preparation by sonication

Difficult to enzymatically digest cross-linked DNA

(Adapted from O'Neill and Turner, Methods, 2003, pp76–82)


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Do you have any ChIP tips you can share with us to help others along?

Contact the Chromatin Team at: chromatin@abcam.com.


Dr. Andy Bannister (Wellcome CR:UK Gurdon Institute, Cambridge, U.K.) and the Abcam Chromatin Team

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