Chromatin Immunoprecipitation (ChIP)

Finding the right antibody for your ChIP experiment is essential. Not all antibodies work for all applications and you need to ensure your antibody works for ChIP and is specific for your target of interest. Some things to consider:

Antibody validation*

If the antibody has been validated for ChIP, then you can use the manufacturer’s guidelines and any published references to guide your experiment. However, in some cases your target of interest has not been tested in ChIP.

If the antibody has been validated in immunoprecipitation (IP), it has a high probability of working in ChIP. Other methods that require the antibody to recognize the target in a native state are also good indicators, such as immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC).

If the antibody has not been validated in ChIP, an excellent indicator is to perform a ChIP-western. Not only does a ChIP-western provide confidence that the antibody pulls down the protein of interest, it can also provide information about specificity. Although, a ChIP-western is labor intensive as the workflow is the same as ChIP through IP step, ChIP-westerns can be performed in parallel to ChIP using a fraction of the IP for ChIP-western and saving the rest for ChIP. After pulling down the protein, instead of eluting the DNA, the proteins are eluted from the beads by boiling and a western blot is performed. Probing the blot with a different antibody to the same target demonstrates that the antibody you chose is recognizing and immunoprecipitating an epitope of the protein of interest. Further, if you have blocking peptides available, specificity can be confirmed as the IP should not work or a significant decrease in efficiency should occur in the presence of the peptide.

Antibody specificity

Specificity of the antibody is a growing and understandable concern, particularly in ChIP-seq. Invitrogen antibodies undergo a two-part testing approach: functional application validation and targeted specificity verification. Functional application validation provides information on whether the antibody works in ChIP. Target specificity verification ensures the antibody is recognizing the target protein of interest. This can be achieved by several methods including the use of knockout and knockdown cell lines, treatment of cells that alters target expression levels, relative expression of the target of interest (if it is differentially expressed), and immunoprecipitation-mass spectrometry (IP-MS). Invitrogen antibodies that have been verified for specificity by one of these techniques have an Advanced Verification badge on our website.

Shearing the chromatin allows the chromatin to be soluble and dictates the resolution of the experiment. The ideal fragment size is 200 to 800 bp. However, chromatin shearing is challenging to control and varies depending upon cell density, extent of crosslinking, and cell type. Thus, it is imperative to be consistent from experiment to experiment and shearing conditions need to be optimized for each cell type.

Enzymatic chromatin shearing

Micrococcal nuclease (MNase) digests unbound DNA and has traditionally been used to map nucleosomes. Enzymatic chromatin shearing does not require specialized equipment, is generally reproducible (although it requires optimization for cell type), and requires minimal hands-on time. One major drawback to MNase is that it is not random and has sequence preferences for digestion of the DNA. Additionally, enzymatic shearing is not a good option for difficult-to-lyse cells as the enzyme will not efficiently enter the cells.

Mechanical chromatin shearing

Sonication is commonly used for chromatin shearing as it produces random fragments. Since sonication uses energy to disrupt the chromatin, it is an ideal choice for hard-to-lyse cells. Like enzymatic shearing, mechanical shearing requires optimization for cell type and unlike enzymatic shearing, sonication requires significant hands-on time.

Determining if your ChIP experiment worked can be quite challenging. Ideally you want to identify a region that you expect to be enriched in your ChIP (i.e., a DNA region where your protein is bound) and a region that you expect to be depleted in ChIP (i.e., a DNA region where your protein is absent). Once these regions are identified, you need to identify and test primers for that region. Primers should amplify a region from 100 to 200 bp and have an efficiency of 90% to 105%.

Sometimes you do not know where your protein will be bound and perform ChIP-seq to learn more about your protein of interest. To provide confidence that your ChIP worked, you can compare the amount of DNA pulled down with and without antibody, with significantly more DNA in the presence of antibody. Also, ChIP-western (see above) can provide confidence that your antibody is pulling down an epitope of the target protein.