There’s a reason chromosome immunoprecipitation (ChIP) continues to be used in labs all over the world. It works well. In fact, there aren’t too many ways 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 yield significant insights into how genes are regulated in their natural context. Although the basic principles of the method are pretty much the same as when it was introduced, new downstream analysis techniques are enabling researchers to get more data than ever out of their ChIP experiments. Many of our readers are chromatin gurus and way more versed in the field than us (that’s fine…we bet they can’t karaoke Guns and Roses like us), but we also know there are just as many researchers out there that are just getting acquainted with ChIP analyses. That’s why we wanted to pass on some useful tips and considerations for ChIP experiments from our friends at Abcam.
The core principle of ChIP is based on selective enrichment of a chromatin fraction containing a specific antigen. Antibodies that recognize a protein or protein modification of interest (e.g. transcription factor, histone modification etc.,) are 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.
A quick note: Variations of ChIP such as MeDIP (5-methylcytosine enrichment) and RIP (Ribonucleic Protein) are becoming increasingly popular in labs studying gene regulation today as well. These methods are discussed elsewhere in EpiGenie.
The ChIP procedure used to be about as fun as a root canal with no anesthesia, but that’s changing with the introduction of optimized commercial kits and streamlined lab protocols. That said, the procedure isn’t bulletproof, so let’s go through some of the areas that can make or break your results.
Upfront processing such as cross-linking of DNA and proteins is often required to stabilize their interactions before analysis. ChIP can be performed in two different ways depending on whether you opt to cross-link your chromatin sample: cross-linking ChIP protocols are often referred to as “X-ChIP,” whereas protocols using native chromatin (no cross-linking) are often termed “N-ChIP.”?
Cross-Linking vs. Au Naturale
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.
- Cross-Linking TIP #1 The further away from the DNA your interaction of interest lies, the less effective ChIP will be without cross-linking. For example, 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. X-ChIP protocols that use cross-linking almost always use formaldehyde, as the links it forms are reversible. UV cross-linking is not appropriate as it is irreversible.
- Cross-Linking TIP #2 Cross-linking is a time-critical procedure and 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. Always perform a time-course experiment to optimize cross-linking conditions.
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 (discussed in Detection).
- Enzymatic Digestion Tip #1 Be sure to aliquot your stock enzyme after purchase and run a new time courses 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 others before it.
- Enzymatic Digestion Tip #2 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.
- X-ChIP Fragmentation Tip Although sonication is typically more effective than enzymatic digestion of fully cross-linked chromatin, enzmatic digestion can work with gentle or incompletely cross-linked samples and will often improve resolution when used in combination with sonication.
Antibodies: The IP in ChIP
Antibodies are the workhorses used in ChIP to capture proteins and the interacting DNA. Considered my many ChIP experts as the most critical component of the process, antibody selection and characterization can have a huge impact on the success/failure of ChIP experiments.?
The specificity of each antibody needs to be well characterized. Antibodies should be tested using ELISA and Western blot (using target and non-target antigens as competitors) to confirm specific epitope recognition.
Western blotting can be used to demonstrate that the correct target has been successfully immunoprecipitated. 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 that recognise multiple antigens in the chromatin fraction should be avoided (or at least this should be considered in the final interpretation). Immunofluorescence can also be used to check that antigen recognition occurs in a more natural context, and can also be combined with competition assays.
WARNING: 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.
Polyclonal or 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.
ChIP Grade Antibodies
In general, if an antibody works in normal immunoprecipitation then it is a good candidate for success in ChIP. Hear what “ChIP Grade” means to various antibody suppliers in our panel discussion with industry experts.
Antibody Control Guidelines
As a positive antibody control for the technique, Histone H3 (tri methyl K4) is a popular positive control to use when ChIPing active genes. As a negative control, use an antibody that recognizes a non-chromatin epitope such as an anti-GFP antibody.?
- Antibody Control 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.
WARNING: 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.
Antibody TroubleShooting Tip
Lots of questions pop up when ChIP experiments aren’t working, like “Why me, why did my PI make me do this experiment?” W can’t answer that, but here’s some feedback to a couple frequently asked questions.
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.
ChIP Downstream Analysis
The downstream analysis techniques have evolved tremendously since ChIP was first introduced. Ten years ago, PCR and gels dominated the scene, while today we see high density arrays (ChIP on Chip) and deep sequencing (ChIP-Seq) leading the way in global analyses.?
ChIP Sample QC
Even if you are thinking about using some of the sexy new approaches that are grabbing the headlines and speaker slots at conferences, you probably want want to use the tried and true methods for some upfront QC measures on your samples before you spend a few weeks on experiments, data acquisition, and analysis of global profiling data.
Certain areas of the genome will purify better than others, and some nucleosomes may re-arrange during enzymatic fragmentation. Try generating some PCR primers to amplify 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.
Major advances in sequencing have made ChIP on Chip and ChIP-Sequencing great options for global profiling, but ChIP qPCR remains a staple for validation as well focused profiling. We’re not going to cover ChIP analysis options in depth here though since they deserve a Method Spotlight to themselves. Stay tuned!