ChIP and ChIP-on-chip Workflow

Chromatin immunoprecipitation (ChIP) experiments are routinely performed to examine histone modifications and genomic DNA sequences bound to specific regulatory proteins. Briefly, in ChIP protein-DNA complexes are crosslinked in vivo, immunoprecipitated, purified, and amplified for gene- and promoter-specific analysis of known targets or for the identification of new target sequences. In a related microarray-based method, ChIP-on-chip, the purified immunoprecipitated DNA is labeled and hybridized to a variety of high resolution microarray types.

Key activities in the ChIP workflow involve the immunoprecipitation and purification steps as well as the detection method. The success of the technique depends, in part, on the ability of the antibody to bind to the target protein after crosslinking. Chip-grade antibodies are available for many proteins of interest. Where not available, an alternative method fuses the proteins to tags such as HA or c-myc - which are recognized by commercially-available antibodies. Magnetic bead-based antibody capture using Dynabeads® has emerged as a leading technique due to the high sensitivity and minimal loss of target protein. Popular detection methods of purified immunoprecipitated DNA involve quantitative PCR (Hecht et al., 1996) and real-time quantitative PCR (Litt et al., 2001).

• MAGnify™ Chromatin Immunoprecipitation System Information Sheet
• Protocols using Invitrogen’s qPCR and qRT-PCR reagents

Since the first published articles to report on successful ChIP-on-chip experiments in 2000 (Ren et al 2000) and 2001 (Lieb et al 2001; Lyer et al 2001), microarray analysis has become a preferred tool for analyzing histone modifications and identifying binding sites for individual transcription factors on a genome wide level (Lee et al 2002).

In addition to the use of self-printed arrays in ChIP-on-chip experiments, commercial array providers have made available mammalian whole genome arrays – including those based on the National Human Genome Research Institute (NHGRI) ENCODE project.

Current topics in chromatin research
New to chromatin research? Learn about intriguing advances.

• Overview of chromatin biology
• Histone modifications, histone variants, and chromatin remodeling
• Overview of DNA methylation

Wu, J. et. al. ChIP-chip Comes of Age for Genomic-wide Functional Analysis. Cancer Research. 2006; 66: 6899-902.

Euskirchen, et. al. Mapping of Transcription Factor Binding Regions in Mammalian Cells by ChIP: Comparison of Array- and Sequencing Based Technologies. Genome Research. 2007; 17: 898-909.

Hecht A., et. al. Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature. 1996; 383(6595): 92-96.

Lyer VR., et. al. Genomic Binding Sites of the Yeast Cell-cycle Transcription Factors SBF and MBF. Nature. 2001; 409(6819): 533-538.

Lee TI., et. al. Transcriptional Regulatory Networks in Saccharomyces cerevisiae. Science. 2002; 298(5594):799-804. 

Lieb JD., et. al. Promoter-specific binding of Rap1 revealed by  genome-wide maps of protein-DNA association. Naure Genetics. 2001; 29(1): 100.

Litt MD., et. al. Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science. 2001; 28: 2453-2455.

Ren B., et. al. Genome-wide location and function of DNA binding proteins. Science. 2000; 290(5500): 2306-2309.