Tips for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful technique to determine protein interactions at particular regions of DNA to map the relative position of chromatin and DNA binding proteins such as histone modifications. Generally, the DNA-protein and protein-protein interactions neighboring chromatin (≈2 Å) in live cells are cross-linked upon treatment with formaldehyde (cross-link ChIP, XChIP) but it can be also performed without cross-linking (native ChIP, NChIP). Following cell lysis, chromatin is sheared by either sonication or enzymatic digestion with micrococcal nuclease (MNase) to generate ∼500 bp DNA fragments. The clarified cell extract is then used for immunoprecipitation (IP) with an antibody that recognizes a target protein, modified peptide (e.g., acetylated, phosphorylated, methylated), or epitope tag. DNA sequences that directly or indirectly cross-link with a given target are selectively enriched in the immunoprecipitated sample. The obtained DNA is de-crosslinked, purified, and analyzed by PCR, quantitative qPCR, labeling, and hybridization to genome-wide or DNA microarrays (ChIP-on-chip), molecular cloning, and sequencing or direct high-throughput sequencing (ChIP-seq). Recent advances on ChIP have simplified the technique in terms of sample requirements, handling and timelines while improving reproducibility and applicability. These are some points to keep in mind when doing ChIP:

• Choosing the right antibody control for ChIP experiments

  As with many other experiments involving antibodies, it is always advisable that you use an isotype control for your antibody to determine the specificity of the observed signal. It is advisable that you perform a mock IP in parallel with an isotype control or with an antibody that is non-related to your protein of interest or the same antibody used in ChIP that is blocked with specific peptide. If working with an epitope tag protein, then you can also do the mock IP with a lysate that doesn’t contain the tagged protein. If multiple antibodies are used with the same chromatin preparation, then a single isotype control is sufficient as long as all the antibodies are from the same species.

• Always use the right antibody

  When performing ChIP assays always make sure that your antibody has been tested for ChIP applications. If a ChIP-validated antibody against your protein of interest is not available, you can also try an antibody that has been tested for IP. Remember that antigen binding can be significantly affected by the loss of epitope accessibility and/or recognition resulting from the cross-linking step. If you suspect this could be a problem, consider using NChIP.

• Make sure to have a chromatin preparation that suits your downstream applications

  When studying transcription factors and other chromatin-associated proteins, including those with weak DNA binding, it is recommended that you use XChIP. Instead, mapping of post-translationally modified histones and histone variants in the genome is better achieved with nChIP.

• Chromatin Fragmentation

  Because the size of DNA fragments (∼500 bp) generated for ChIP constitute a critical parameter in achieving good mapping resolution and efficient solubilization upon cell lysis, it is essential to estimate fragmentation by 1.2% agarose gel electrophoresis following DNA purification. This is a variable highly dependent on the extent of cross-linking that would otherwise generate larger, less soluble fragments when done excessively. Thereby, this confirmation step should be performed every time that fixation conditions are changed. Since it is not possible to check the efficiency of shearing in this way with less than ∼100,000 cells, it is recommended to optimize the shearing on a higher amount of cells before doing ChIP on actual samples of fewer cells. If using MNase, adjusting the concentration of the enzyme will result in different fragment sizes.

• Optimize the ratio of antibody to chromatin used in your IP

  Performing preliminary experiments to empirically determine the lowest antibody concentration that depletes >90% of the protein of interest from the extract will help you to improve the IP step. These experiments should be analyzed by Western blot after trying different dilutions of antibody with chromatin devoid of cross-linking. If working with abundant proteins, 1–2 µg of antibody per ChIP should be enough whereas low abundant targets might require as much as 10 µg. This will help to optimize the efficiency and specificity of the enrichment in your actual ChIP experiment, especially if using cross-linked chromatin that is reported to exhibit ∼50% reduction in IP efficacy as a result of epitope modification.

• Preserving post-translational modifications

  When mapping the location of PTM histones and other chromatin-associated proteins, you might see a several-fold enhancement in the enrichment efficiency by using specific inhibitors that prevent the degradation of the target modification. This is particularly relevant for some labile post-translational modifications including histone acetylations and phosphorylations that can be efficiently preserved by using histone deacetylase and phosphatase inhibitors in all the solutions used before fixation and thereafter.

• Confirm protein enrichment of your target sequence

  When working with your ChIP data it is always advisable to compare the obtained enrichment of an immunoprecipitated genomic region to several other unrelated regions in the same experiment. Those irrelevant regions should generate a typical background of ∼0.025%–0.05% for IP efficiency and resulting in between >5-fold–100-fold enrichment for regions bound by the protein of interest.

• False negatives

  Depending on how the protein of interest is bound to DNA and other associated proteins, it is possible that the target epitope is not always accessible to the antibody during IP, resulting in false negatives. This might be the case in those ChIP experiments where no enrichment is observed for any genomic region. The use of a polyclonal antibody or an epitope tag can help to diminish this problem by either increasing the possibility of recognizing more epitopes or using an epitope that is expected to be rather inert.

• Shorten the time for ChIP

  One of the drawbacks of traditional ChIP is the length of the whole procedure. Several advances in this subject have led to time optimization of some stages, including combining distinct steps for cross-link reversal, proteinase K digestion, and DNA elution into a single 2-hour step and shortening the antibody incubation time during IP by 15 minutes when performed in an ultrasonic bath.

• Always verify the quality of your ChIP DNA

  Verify that a minimum of 5-fold enrichment of target DNA is measured in your ChIP DNA by either PCR or qPCR when compared to the isotype control and remember that as with any other qPCR assay, the efficiency of the primers used is critical. For a problematic PCR, you can also include a PCR on genomic DNA as a positive control. A frequently recommended starting dilution for the ChIP sample and controls is 1:100.