Additionally, interacting proteins may mask the epitope of the protein of interest. Finally, the ChIP technique is extremely dependent on the quality and specificity of the antibody employed and may not discriminate between different DNA-binding protein isoforms.
While the principle of the ChIP technique is quite simple, the actual execution of a ChIP experiment can be rather complex. Here are some important factors to consider when planning or trouble-shooting ChIP assays:. The quality of the chromatin generated in ChIP can make the difference between an uninterpretable result and a quality data point.
In particular, sonication is typically used to break chromatin down into shorter fragments, but sonicating for too long or too harshly can lead to denatured protein, or protein that is dissociated from DNA or whose antibody epitope has been destroyed. This step requires considerable optimization. Four key variables to consider are cell density, power, time and number of cycles. Be sure to keep chromatin on ice at all times, and limit pulses to under 30 seconds at a time to prevent protein denaturation from overheating.
While antibodies for western blotting can recognize denatured protein, ChIP requires recognition of the tertiary structure of a protein of interest and of an exposed epitope. Another consideration is the choice between monoclonal or polyclonal antibodies. While monoclonal antibodies have higher batch-to-batch consistency, they recognize a single epitope, which may be masked through binding to other proteins or buried within the tertiary structure of the protein.
Polyclonal antibodies on the other hand represent a population of a number of different antibodies that recognize different epitopes. This reduces the probability that all specific epitopes are masked due to cross-linking and increases the chance of a positive result. Alternatively, if no good antibody candidates exist, it is possible to tag the protein of interest with a Myc, His or GST tag, for example, and use antibodies targeting the tag of choice.
It is also important to optimize the number of cells for your particular ChIP experiment. This will depend on the abundance of the target being studied. Histone modifications are quite abundant, and ChIPping for these marks generally requires less starting material than probing for transcription factors, which are significantly scarcer. Prolonged cross-linking can reduce antigen availability and efficiency of the chromatin-shearing step.
Excessive cross-linking may mask the epitope that is recognizable by your antibody, leading to a reduction in protein that can be pulled down. As for any experiment, controls are indispensable in ChIP to calculate enrichment in protein binding and to interpret the significance of a result.
The following controls are recommended:. Let me know in the comment section below if you have specific questions regarding your ChIP experiment. Happy researching! Platform Science Culture Perspectives. Site Search Desktop. How do I cross-link? Use formaldehyde, as the links it forms are reversible.
UV cross-linking is not appropriate as it is irreversible. It should be noted that alternative cross-linkers to formaldehyde do exist—these may be useful if the researcher need to cross-link over various intermolecular distances. Can I cross-link too much? 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 several issues including 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. Always perform a time-course experiment to optimize cross-linking conditions.
We would suggest cross-linking the samples for 2—30 min. Glycine is added to quench the formaldehyde and terminate the cross-linking reaction. To further aid DNA purification, cross-links between proteins and DNA are disrupted by treatment with proteinase K, which cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids.
Fragmentation of the chromatin is required to make interactions accessible to antibody reagents. To fragment chromatin, you can either sonicate it or digest it using micrococcal nuclease.
The method you choose will largely depend upon the type of ChIP experiment being performed. Whatever method you are using be sure to run a fragmentation time course every time you set up an experiment. 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 effects on the enzyme accessing its target. However, certain chromatin binders, such as transcription factors, often bind inter-nucleosomal DNA so purified mono-nucleosomes are not suitable.
Additionally, 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. Enzymatic cleavage will not produce random sections of chromatin. Micrococcal 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. 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. 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.
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, although in practice this is rarely observed.
The fragments created by sonicating, which average — 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. 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 as it results in a decrease of energy transfer within the solution and will decrease the sonication efficiency. Avoid multiple cycles of freeze thaw. Make sure the antibody works in ChIP. If available, use an antibody that has been fully characterized and labeled as ChIP-grade. 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.
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.
To test whether an uncharacterized antibody can ChIP, you can perform a ChIP with the antibody followed by a western blot with the same antibody. 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. Antibodies for histone modifications need to be thoroughly tested for specificity, e. Polyclonal vs. 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.
However, monoclonal antibodies usually have higher batch to batch consistency. What antibody controls could I use? As a positive antibody control for the technique, 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 e. When analyzing histone modifications, you need to normalize to histone content. This can be done with the anti-H3 antibody ab 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. 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 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: it may be necessary to further optimize the cross-linkage time course.
What concentration of antibody should I use in my ChIP experiment? 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.
Even if the antibody is able to immunoprecipitate the protein of interest in formaldehyde fixed chromatin this does not mean that the ChIP experiment has worked, as it is possible that your protein of interest is not cross-linked to the DNA.
If high background is observed, additional washes may be needed.
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