Chromatin Immunoprecipitation is a powerful tool to study protein:DNA complexes and can be used to map transcription factor binding sites or to study epigenetic modifications. In this assay, chromatin bound proteins are crosslinked and the DNA is fragmented. Next, the sheared DNA:protein complexes are incubated with an antibody against the protein-of-interest and immunoprecipitated to enrich for bound DNA. Finally, the crosslinks are reversed and the DNA is eluted for analysis. Purified DNA can analyzed using qPCR, Next-Generation Sequencing, ChIP-on-chip analysis, etc.

Click on the steps below to learn more about how to optimize your ChIP assays.

 

 

Fixation and Chromatin Isolation

 

 

 

 

 

 

 

 

 

DNA Shearing

 

 

 

 

 

 

 

 

 

Immunoprecipitation

 

 

 

 

 

 

 

 

 

 

 

Downstream DNA analysis

 

 

Fixation and Chromatin Isolation
A successful ChIP experiment is dependent on high quality chromatin. Chromatin quality can be affected by lysis, fixation, and shearing conditions. As chromatin can degrade very quickly, it should be kept on ice throughout the experiment, and freeze/thaw cycles should be avoided.

Fixation
Formaldehyde is commonly used to reversibly cross-link proteins bound to DNA. For consistent crosslinking results, ensure that the fixation solution is made fresh and is methanol-free. For larger complexes or indirect protein interactions, formaldehyde can be used in combination with other cross-linkers such as EGS or DSG. However, other fixation methods, such as UV cross-linking are not reversible and are not suitable for ChIP applications.

Fixation conditions must be optimized for each experiment as over-fixing your samples may make them resistant to subsequent lysis and shearing steps. Additionally, over-fixation will make it more difficult to reverse cross-links and interfere with downstream analysis.

Cell lysis
Lysis buffers used for ChIP experiments contain detergents which can precipitate out of solution at cold temperatures. As such, lysis buffers should be warmed to room temperature to ensure precipitates are fully dissolved before use. Excess detergent in the lysis buffer may inhibit antibody binding during immunoprecipitation, so lysis buffer volume and composition must be optimized.

 

 

DNA Shearing
Chromatin can be sheared using two methods: sonication or enzymatic digestion. Regardless of the method chosen, chromatin should be sheared to fragments of 150-900 base pairs. Regardless of the method chosen, chromatin should be sheared to fragments of 100-900 base pairs. If the chromatin is over-sheared, it risks damaging the epitope bound by the protein or may prevent qPCR primers from recognizing the DNA fragments. If chromatin is under-sheared, it may cause an increase in non-specific binding.

Sonication
Sonication is a mechanical shearing of chromatin using ultra sonic sound waves. The use of this approach is advantageous as it results in random fragmentation of the DNA and is suitable for difficult to lyse cell types or those in which cross-linking has made it difficult for enzymes to access its sites. However, this method requires expensive equipment and can potentially damage epitopes. Typically, sonication for 30 sec on/off for a total of 15 mins works for shearing, though this must be optimized for each cell type.

Enzymatic Digestion
Enzymatic Digestion typically relies on the use of micrococcal nuclease for digestion. It is a milder treatment and it does not require the use of expensive equipment. However, the fragmentation will exhibit sequence bias. In this case, it is critical to optimize the concentration of enzyme used and adjust the digestion times for optimal shearing.

 

 

Antibody Selection
Choosing an antibody that will work well in ChIP applications and is specific for the target protein is critical for a successful ChIP experiment. As the antibody must recognize the native form of the protein, antibodies used in other applications may not work in ChIP experiments. We recommend using a ChIP-validated antibody in addition to control antibodies such as an RNA polymerase II antibody.

The amount of antibody used is critical for any ChIP experiment. If there is too much antibody, it can lead to non-specific binding whereas too little antibody will not be able to bind all of the chromatin-bound protein resulting in misrepresentation of the antibody enrichment. The end-user can consult the product datasheet for a recommend usage of their ChIP-validated antibody, but the optimal concentration of each antibody should be determined empirically for each experiment.

Immunoprecipitation
Immunoprecipitation of chromatin-bound protein can be performed with Protein A, G, or A/G magnetic or agarose beads. In general, Protein A will exhibit greater specificity for rabbit polyclonal antibodies whereas protein G exhibits a broader specificity. Alternatively, our Go-ChIP-Gradeā„¢ kits feature columns containing a solid-phase scaffold developed by Chromatrap for immunoprecipitation, which provides increased cell surface area for antibody binding and reduces nonspecific signals.

 

 

Downstream Analysis and Controls
After the cross-links have been reversed, the purified DNA can be analyzed using qPCR or used to generate libraries for Next Generation sequencing. For qPCR analysis, we recommend designing primers to amplify a 100-250 bp region around the gene or binding site of interest to test alongside a positive control primer targeting a known binding site.

In addition to using positive and negative control primers, an input sample should be saved prior to performing the immunoprecipitation so that the enriched DNA can be normalized to the total amount of chromatin used in the experiment. Typically, 1-5% of the starting chromatin is used.

To calculate the percent signal relative to input, use the following calculation:

  1. Adjust Ct of Input sample to 100%.
         For a 5% Input control, subtract 4.32 (log2 of 20) cycles from the experimentally determined Ct value.
  2. Calculate the percent input of each sample including isotype controls.
         Use the equation % input = 100*2^(Adjust Input Ct- Sample Ct)