Chromatin immunoprecipitation (ChIP) assays begin with covalent stabilization of protein–DNA complexes. Many protein–DNA interactions are transient, and involve multi-protein complexes to orchestrate biological function. In vivo crosslinking covalently stabilizes protein–DNA complexes.
In vivo crosslinking is traditionally achieved with formaldehyde but can be combined with other crosslinkers such as EGS and DSG. Formaldehyde crosslinking is ideal for two molecules which interact directly. However, formaldehyde is a zero-length crosslinker, limiting its functionality. For higher order interactions, longer crosslinkers such as EGS (16.1 Å) or DSG (7.7 Å) can trap larger protein complexes with complex quaternary structure.
Researchers often use a combination of crosslinkers to trap protein–DNA interacting partners. These crosslinkers permeate directly into intact cells to effectively lock protein–DNA complexes together, allowing even transient interaction complexes to be trapped and stabilized for analysis.
This diagram depicts covalent protein–DNA interactions within the nucleus.
This updated overview of the ChIP procedure includes additional detail about primary antibody selection (i.e., ChIP-validated antibodies). The application note also describes and provides examples of chromatin immunoprecipitation (ChIP) as a technique for studying epigenetics, as it allows researchers to capture a snapshot of specific protein–DNA interactions.
The lysis stage extracts the crosslinked protein–DNA complexes from cells or tissue and brings them into solution. At this stage, cellular components are liberated by dissolving the cell membrane with detergent-based solutions. Because protein–DNA interactions occur primarily in the nuclear compartment, removing cytosolic proteins can help reduce background and increase sensitivity. The presence of detergents or salts will not affect the protein–DNA complex, as the covalent crosslinking achieved in step one will keep the complex stable throughout the ChIP procedure.
Mechanical lysis of cells is not recommended, as it can result in inefficient nuclear lysis. Reagents such as the Thermo Scientific Pierce Chromatin Prep Module, which isolate the nuclear fraction from other cellular components, are used to eliminate background signal and enhance sensitivity.
This diagram illustrates protein–DNA interactions in a cell that has undergone lysis.
Learn more about how to desalt, buffer exchange, concentrate, and/or remove contaminants from protein samples, immunoprecipitation and other protein purification and clean up methods using various Thermo Scientific protein biology tools in this 32-page handbook.
The extraction step yields all nuclear material, which includes unbound nuclear protein, full length chromatin and the crosslinked protein–DNA complexes. In order to analyze protein-binding sequences, the extracted genomic DNA must be sheared into smaller, workable pieces. DNA fragmentation is usually achieved either mechanically by sonication or enzymatically by digestion with micrococcal nuclease (MNase).
Ideal chromatin fragments can range from 200 to >1000 bp; however, DNA shearing is one of the most difficult steps to control. Sonication provides truly randomized fragments, but limitations include the requirement of dedicated machinery that may need tuning, difficulty in maintaining temperature during sonication, and extended hands-on time and extensive optimization steps. Enzymatic digestion with micrococcal nuclease is highly reproducible and more amendable to processing multiple samples, but can lead to variability due to changes in enzyme activity and the enzyme having higher affinity for inter-nucleosome regions.
This illustration represents interactions between protein–DNA complexes and depicts sheared genomic DNA.
To isolate a specific modified histone, transcription factor or co-factor of interest, ChIP-validated antibodies are used to immunoprecipitate and isolate the target from other nuclear components. This step selectively enriches for the protein–DNA complex of interest and eliminates all other unrelated cellular material.
Selection of the appropriate antibody is a critical parameter to successful ChIP assays. For mammalian samples, numerous ChIP-grade antibodies are available that are validated for this procedure. For target proteins for which qualified antibodies are unavailable, fusion proteins such as HA, myc or GST can be expressed in the biological sample, and then antibodies against the affinity tags can be used to immunoprecipitate the target.
The antibody–protein–DNA complex is affinity-purified using an antibody-binding resin such as immobilized protein A, protein G or protein A/G. For biotinylated antibodies, immobilized streptavidin or immobilized Thermo Scientific NeutrAvidin Protein can also be used. For reduced background, it is necessary to block antibody-binding beads with a combination of nucleic acid and protein blocking buffers such as salmon sperm DNA and a generic protein source. The volume of beads used in each ChIP sample can also influence background, as the increase in bead volume increases nonspecific binding.
This diagram shows target proteins that are immunoprecipitated along with crosslinked nucleotide sequences. Subsequently, the DNA is removed, identified by PCR, sequenced, applied to microarrays, or analyzed in some other fashion.
Enrichment of DNA bound to the protein of interest is the goal for chromatin immunoprecipitation. DNA levels can be determined by agarose gel electrophoresis or more commonly by quantitative polymerase chain reaction (qPCR). Before the specific DNA products of a chromatin IP assay can be amplified and measured, the crosslinks between protein and DNA must be reversed. This is typically done through extensive heat incubations or through digestion of the protein component with proteinase K.
Proteinase K cleaves at the carboxy side of aliphatic, aromatic or hydrophobic residues. Because of its broad specificity, proteinase K is often used for removing proteins from DNA or RNA preparations. Additionally, proteinase K digestion eliminates nucleases from the purified DNA, which prevents degradation. To separate DNA from protein fragments, phenol-chloroform is used in conjunction with a standard DNA purification method. Alternatively, spin columns designed to purify nucleic acid material from complex biological samples may be used.
The illustration shows DNA liberated after crosslinking reversal and DNA clean-up steps.
Despite being amplification techniques, qPCR procedures are sufficiently accurate to enable measurement of target protein–DNA levels in different experimental conditions. There is a direct correlation between the amounts of immunoprecipitated complex and bound DNA.
A hallmark of ChIP is the ability to quantitate the purified DNA products with quantitative PCR (qPCR).
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