Chromatin immunoprecipitation (ChIP) is a technique used in epigenetic research that takes a snapshot of protein-DNA interactions. While selecting the right antibody is critical, all the steps in the ChIP process are important in order to obtain great results. This technique makes use of a variety of molecular biology and proteomic methods.
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:
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.
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.
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.
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.
Consider these five proven steps to help ensure meaningful results:
This first step is time-sensitive and requires optimization. In vivo covalent crosslinking stabilizes protein-DNA interactions for analysis at a specific point in time. Formaldehyde is commonly used for cross-linking, while glycine is added to quench the reaction. The crosslinkers permeate into the intact cells and lock the protein-DNA complexes together, enabling analysis. The crosslinkers keep the complexes steady throughout the procedure, but must be reversible to be used for ChIP. For some very stable protein-DNA interactions, crosslinking is not necessary.
Next, the cell membrane is permeabilized with lysis solutions, the cellular components are liberated, and the protein-DNA complexes become soluble. Cytosolic proteins are removed to reduce background signal and increase sensitivity.
Note: The ChIP assay can be stopped at this point. After crosslinking, quenching, and washing the cell pellet, the lysate can be stored at -80°C.
The nuclear material must be fragmented, and this fragmentation is key for good ChIP resolution, ideally resulting in fragment sizes between 200 and 800 base pairs. Shearing is one of the most difficult steps to control. It can be achieved by sonication and/or nuclease/enzymatic digestion, each with limitations and benefits. Sonication requires significant hands-on time, but is ideal for hard-to-lyse cells, while enzymatic digestion is relatively hands-off, accommodates multiple sample processing, but is not random. Both methods require optimization for individual cell lines.
Note: ChIP can be stopped at this point. After shearing/digestion of the chromatin, the sample can be stored at -80°C. Avoid repeated freeze/thaw.
Antibodies are used to capture the protein of interest in the protein-DNA complex, and they are a crucial factor in a successful experiment. Antibodies immunoprecipitate and isolate the protein from the nuclear components, eliminating the unrelated cellular material.
Polyclonal antibodies have the advantage of recognizing multiple epitopes, but there is more lot-to-lot variation and they are a limited resource. Our recombinant monoclonals practically eliminate lot-to-lot variation and are not a limited resource, but they can be limited in the protein conformations they recognize. Our recombinant oligoclonals may be the best of both worlds as they are a pool of recombinant monoclonals and so can recognize multiple epitopes with limited lot-to-lot variability and are a renewable resource.
If no antibodies are available for certain targets, protein fused to affinity tags (HA, Myc, His, etc.) can be expressed in the samples, then immunoprecipitated using antibodies against the affinity tags.
Purification of the resulting antibody-protein-DNA complex is accomplished using antibody-binding beads. The bead-antibody-protein-DNA complex is washed extensively after incubation, purified, and the protein-DNA complex eluted.
Now that chromatin fractions containing your protein of interest have been isolated, the crosslinks need to be reversed and the DNA purified. The reversal of crosslinking is typically done through extensive heat incubation and/or digestion of the protein component.
In order to obtain a purer DNA sample, treatment with RNase A is recommended. To purify the DNA from the remaining proteins, phenol:chloroform extraction or spin columns for DNA purification should be used.
Note: ChIP can be stopped at this point. After crosslink reversal and/or DNA purification, samples can be stored at -20°C.
At this step, the purified DNA products are quantitated by qPCR, which can be achieved using the NanoDrop spectrophotometer, Qubit Fluorometer, or other spectrophotometric method. Using qPCR, you can determine if your protein is present at specific loci. SYBR green fluorescent dye is the most widely used DNA-based qPCR chemistry. SYBR green is fluorescent only when bound to double-stranded DNA (dsDNA), and the fluorescence is proportional to the amount of dsDNA. This allows you to quantitate the extent of enrichment of the target of interest at certain regions of DNA. Optimization of primer sets is required at this step to obtain accurate quantification.
The NanoDrop Spectrophotometer provides a very simple way to quantitate DNA as well as assess for any solvent contaminants using the 230/260 nm absorbance ratio.
The Qubit Fluorometer can accurately quantitate low levels of purified DNA and be used to normalize samples prior to qPCR or for use in sequencing applications.
Recommended products for DNA quantitation:
This first step is time-sensitive and requires optimization. In vivo covalent crosslinking stabilizes protein-DNA interactions for analysis at a specific point in time. Formaldehyde is commonly used for cross-linking, while glycine is added to quench the reaction. The crosslinkers permeate into the intact cells and lock the protein-DNA complexes together, enabling analysis. The crosslinkers keep the complexes steady throughout the procedure, but must be reversible to be used for ChIP. For some very stable protein-DNA interactions, crosslinking is not necessary.
Next, the cell membrane is permeabilized with lysis solutions, the cellular components are liberated, and the protein-DNA complexes become soluble. Cytosolic proteins are removed to reduce background signal and increase sensitivity.
Note: The ChIP assay can be stopped at this point. After crosslinking, quenching, and washing the cell pellet, the lysate can be stored at -80°C.
The nuclear material must be fragmented, and this fragmentation is key for good ChIP resolution, ideally resulting in fragment sizes between 200 and 800 base pairs. Shearing is one of the most difficult steps to control. It can be achieved by sonication and/or nuclease/enzymatic digestion, each with limitations and benefits. Sonication requires significant hands-on time, but is ideal for hard-to-lyse cells, while enzymatic digestion is relatively hands-off, accommodates multiple sample processing, but is not random. Both methods require optimization for individual cell lines.
Note: ChIP can be stopped at this point. After shearing/digestion of the chromatin, the sample can be stored at -80°C. Avoid repeated freeze/thaw.
Antibodies are used to capture the protein of interest in the protein-DNA complex, and they are a crucial factor in a successful experiment. Antibodies immunoprecipitate and isolate the protein from the nuclear components, eliminating the unrelated cellular material.
Polyclonal antibodies have the advantage of recognizing multiple epitopes, but there is more lot-to-lot variation and they are a limited resource. Our recombinant monoclonals practically eliminate lot-to-lot variation and are not a limited resource, but they can be limited in the protein conformations they recognize. Our recombinant oligoclonals may be the best of both worlds as they are a pool of recombinant monoclonals and so can recognize multiple epitopes with limited lot-to-lot variability and are a renewable resource.
If no antibodies are available for certain targets, protein fused to affinity tags (HA, Myc, His, etc.) can be expressed in the samples, then immunoprecipitated using antibodies against the affinity tags.
Purification of the resulting antibody-protein-DNA complex is accomplished using antibody-binding beads. The bead-antibody-protein-DNA complex is washed extensively after incubation, purified, and the protein-DNA complex eluted.
Now that chromatin fractions containing your protein of interest have been isolated, the crosslinks need to be reversed and the DNA purified. The reversal of crosslinking is typically done through extensive heat incubation and/or digestion of the protein component.
In order to obtain a purer DNA sample, treatment with RNase A is recommended. To purify the DNA from the remaining proteins, phenol:chloroform extraction or spin columns for DNA purification should be used.
Note: ChIP can be stopped at this point. After crosslink reversal and/or DNA purification, samples can be stored at -20°C.
At this step, the purified DNA products are quantitated by qPCR, which can be achieved using the NanoDrop spectrophotometer, Qubit Fluorometer, or other spectrophotometric method. Using qPCR, you can determine if your protein is present at specific loci. SYBR green fluorescent dye is the most widely used DNA-based qPCR chemistry. SYBR green is fluorescent only when bound to double-stranded DNA (dsDNA), and the fluorescence is proportional to the amount of dsDNA. This allows you to quantitate the extent of enrichment of the target of interest at certain regions of DNA. Optimization of primer sets is required at this step to obtain accurate quantification.
The NanoDrop Spectrophotometer provides a very simple way to quantitate DNA as well as assess for any solvent contaminants using the 230/260 nm absorbance ratio.
The Qubit Fluorometer can accurately quantitate low levels of purified DNA and be used to normalize samples prior to qPCR or for use in sequencing applications.
Recommended products for DNA quantitation:
*The use or any variation of the word "validation" refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.
For Research Use Only. Not for use in diagnostic procedures.
