ChIP Antibodies

Chromatin immunoprecipitation (ChIP) is an antibody-based technique for studying protein-DNA interactions in cells or tissues. It can reveal how such interactions influence critical processes like gene transcription, DNA repair, and cell cycle progression, and how their dysregulation is involved in disease pathogenesis. Like immunoprecipitation (IP), ChIP uses antibodies that have been immobilized on the surface of functionalized beads for protein pulldown. However, because ChIP specifically captures DNA-binding proteins such as histones, transcription factors, and cofactors in combination with their DNA targets, the protocol differs between the two methods. A typical ChIP experiment begins with crosslinking to stabilize protein-DNA interactions, which are generally short-lived. Formaldehyde is commonly used, although it must be supplemented with an additional crosslinker (e.g., disuccinimidyl glutarate) when capturing multi-protein complexes. A variation of ChIP, known as native ChIP, omits the crosslinking step and may thus improve target recognition by antibodies (by avoiding epitope masking). Yet because native ChIP risks dissociation of protein-DNA complexes, it is mainly reserved for studying histones (which are tightly bound to the DNA). Next the cells are lysed with a detergent-based solution to release the nuclear contents. The genomic DNA is then solubilized by shearing it into smaller fragments, either by sonication (incompatible with native ChIP) or by enzymatic digestion with a micrococcal nuclease (MNase). Following this, a ChIP grade antibody is used to isolate the target of interest through affinity purification with appropriate beads. If a ChIP grade antibody is not available, it may be worth testing an antibody that has been validated for IP, especially if trial size antibodies are available. The types of beads used for ChIP are either agarose or magnetic, and are usually functionalized with Protein A, Protein G, or Protein A/G (a recombinant fusion protein that contains binding domains from both proteins). Streptavidin coated beads are also widely used when working with biotinylated antibodies. To prevent non-specific binding, the beads should be blocked with DNA and a generic protein source. Often, salmon sperm DNA is combined with bovine serum albumin (BSA) for blocking purposes. Once the protein-DNA complex has been captured from solution and eluted from the beads (using detergent and heat), the DNA must be isolated and purified. DNA isolation is achieved by using high temperature and high salt to reverse the crosslinks, and treating with proteinase K to digest the bound proteins and any primary antibodies used for target capture. Purification is based on traditional phenol-chloroform extraction and ethanol precipitation or may instead use a column-based DNA purification kit. Lastly, the purified DNA is analyzed using one of several techniques. These include ChIP-PCR and ChIP-qPCR, which report protein binding to a known subset of target loci and are sufficient for most research applications, and ChIP-chip and ChIP-seq, which are used for genome-wide analysis. The main difference between ChIP-chip and ChIP-seq is that ChIP-chip uses DNA microarrays for analyzing enriched DNA fragments while ChIP-seq uses next-generation sequencing (NGS) technology to align the purified DNA with previously annotated whole genomes.