Overview of Chromatin immunoprecipitation (ChIP)
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The earliest forms of chromatin immunoprecipitation (ChIP) appeared in the early 1980s to study the distribution of RNA polymerases on bacterial genes1. Since this pioneering work, the method of ChIP has evolved and expanded into many forms to study how proteins interact with DNA in vivo in the context of a cell.
What is ChIP?
ChIP is a technique that is based on using antibodies to pull down or immunoprecipitate specific proteins and their associated DNA. DNA associated with a protein of interest is isolated and analyzed. Post-ChIP analysis of DNA is achieved by a number of methods, some of which include polymerase chain reaction (-PCR), microarray chip hybridization (-chip), and next generation sequencing (-seq).
How Does It Work?
Enriched DNA is amplified and labeled with a fluorescent probe and hybridized to a microarray chip
ChIP typically begins with live cells grown in culture. The cells are fixed with formaldehyde to crosslink proteins to their interacting DNA and the cells are lysed to release the proteins and associated DNA. The DNA is sheared either by sonication or nuclease digestion and the antibody against a specific protein of interest is added. The antibody-protein-DNA immunocomplex is then isolated by adsorption to protein-A or -G agarose beads. Immunoprecipitated DNA is eluted from the beads and the formaldehyde cross-links are reversed. After which the DNA is then extracted, precipitated, and solubilized for analysis by one of a number of methods2. The most common methods for analyzing the immunoprecipitated DNA include:
- ChIP-PCR – This method examines DNA by PCR using primers specific to a gene. It is considered a biased analysis and must presume binding of factors to specific DNA sequences2.
- ChIP-chip – This method provides an unbiased survey of immunoprecipitated DNA via hybridization to whole genome microarrays. Enriched DNA is amplified and labeled with a fluorescent probe and hybridized to a microarray chip2.
- ChIP-seq – This method uses next generation sequencing to analyze DNA in an unbiased genome-wide manner. It was developed to overcome limitations of the ChIP-ChIP method3. Immunoprecipitated DNA fragments are sequenced, and enriched regions are compared with input reads and detected as "peaks." The enriched sequences are mapped back to the reference genome to provide sequences bound by the protein of interest4.
ChIP has become a mainstream assay in genomics and epigenetics. It has been used extensively to identify DNA elements and proteins that regulate transcription and to study how histone modifications influence gene expression. The ability to analyze gene regulation and genome-wide epigenetic changes in human cancer and other diseases will lead to improved understanding of the role abnormal gene transcription plays in the development of disease. Furthermore, knowledge of the state of chromatin in this context will advance the development of valuable diagnostic tools and disease therapies.
Bethyl Laboratories sells high quality ChIP antibodies. These products have recently been used to study:
- The formation of gene fusions and how these fusions contribute to aberrant transcriptions and prostate cancer5
- Transcriptional regulators related to neural synapsis and the development of memories6
- How the suppression of miRNAs can impact transcription and lead to unchecked cell differentiation7
- How a protein regulates the cell cycle through the transcriptional regulation of miRNA8
- The role of an RNA processing enzyme in the transcription cycle of Pol II9
Hundreds of Bethyl antibodies are validated for use in chromatin immunoprecipitation. The complete list is here:
1. Gilmour DS, Lis JT. 1984. Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc Natl Acad Sci USA. July;81(14):4275–4279.
2. Das PM, Ramachandran K, vanWert J, Singal R. 2004. Chromatin immunoprecipitation assay. BioTechniques. Dec;37(6):961–969.
3. Barski A, Cuddapah S, Cui K, Roh T-Y, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K. 2007. High-resolution profiling of histone methylations in the human genome. Cell. May 18;129(4):823–837.
4. Nakato R, Shirahige K. 2017. Recent advances in ChIP-seq analysis: from quality management to whole-genome annotation. Briefings in Bioinform. Mar 1;18(2):279–290.
5. Metzger E, Willmann D, McMillan J, Forne I, Metzger P, Gerhardt S, Petroll K, von Maessenhausen A, Urban S, Schott AK, et al. 2016. Assembly of methylated KDM1A and CHD1 drives androgen receptor-dependent transcription and translocation. Nat Struct Mol Biol. Feb;23(2):132-139. [Bethyl antibody used: CHD1 Antibody (A301-218A)]
6. Korb E, Herre M, Zucker-Scharff I, Darnell RB, Allis CD. 2015. BET protein Brd4 activates transcription in neurons and BET inhibitor Jq1 blocks memory in mice. Nat Neurosci. Oct;18(10):1464-1473. [Bethyl antibodies used: BRD2 Antibody (A302-583A) and BRD3 Antibody (A302-368A). Please note that these antibodies have not been validated by Bethyl for use in this application and as such Bethyl cannot guarantee results as published in this paper.]
7. Tajima K, Yae T, Javaid S, Tam O, Comaills V, Morris R, Wittner BS, Liu M, Engstrom A, Takahashi F, et al. 2015. SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes. Nat Commun. Sep 23;6:8257. [Bethyl antibody used: MLL1 Antibody (A300-374A).]
8. Tajima K, Yae T, Javaid S, Tam O, Comaills V, Morris R, Wittner BS, Liu M, Engstrom A, Takahashi F, et al. 2015. SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes. Nat Commun. Sep 23;6:8257. [Bethyl antibody used: hSET1 Antibody (A300-289A)]
9. Sansó M, Levin S, Lipp J, Wang V, Greifenberg A, Quezada E, Ali A, Ghosh A, Larochelle S, Rana T, et al. 2016. P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates. Genes Dev. Jan 1; 30(1): 117–131. [Bethyl antibody used: XRN2 Antibody (A301-103A). Please note that this antibody has not been validated by Bethyl for use in this application and as such Bethyl cannot guarantee results as published in this paper.]